US008551476 B2

(12) United States Patent (10) Patent No.: US 8,551,476 B2 Mi et al. (45) Date of Patent: *Oct. 8, 2013

(54) SP35 ANTIBODIES AND USES THEREOF 5,413,923 A 5/1995 Kucherlapati et al. 5,427,908 A 6/1995 Dower et al. (75) Inventors: Sha Mi, Belmont, MA (US); R. Blake 39 A 3. R al Pepinsky, Arlington, MA (US); Zhaohui 5.530,101 A 6/1996 St. I. Shao, Brookline, MA (US); Christilyn 5,545,806 A 8/1996 Lonberg et al. Graff, Cambridge, MA (US) 5,565,332 A 10/1996 Hoogenboom et al. 5,565,335 A 10/1996 Capon et al. (73) Assignee: Biogen Idec MA Inc., Cambridge, MA 5,569,825 A 10/1996 Lonberg et al. US 5,571,698 A 11/1996 Ladner et al. (US) 5,574,009 A 11/1996 Cohen et al. 5,580,717 A 12/1996 Dower et al. (*) Notice: Subject to any disclaimer, the term of this 5,585,089 A 12/1996 Queen et al. patent is extended or adjusted under 35 5,589,369 A 12/1996 Seidman et al. U.S.C. 154(b) by 1381 days. 5,625,126 A 4/1997 Lonberg et al. 5,633,425 A 5/1997 Lonberg et al. This patent is Subject to a terminal dis- 5,658,570 A 8, 1997 Newman et al. claimer. 5,658,727 A 8, 1997 Barbas et al. 5,661,016 A 8/1997 Lonberg et al. 5,693,761 A 12/1997 Queen et al. (21) Appl. No.: 11/892,036 5,693,762. A 12/1997 Queen et al. 5,693,780 A 12/1997 Newman et al. (22) Filed: Aug. 17, 2007 5,698,426 A 12/1997 Huse 5,725,859 A 3, 1998 Omer (65) Prior Publication Data 5,733,743 A 3, 1998 Johnson et al. US 2009/0017039 A1 Jan. 15, 2009 5,750,753 A 5/1998 Kimae et al. (Continued) Related U.S. Application Data FOREIGN PATENT DOCUMENTS (63) Continuation of application No. EP O 154316 B1 9, 1989 PCT/US2006/026271, filed on Jul. 7, 2006. EP O 401 384 A1 12, 1990 (60) Provisional application No. 60/697.336, filed on Jul. 8, (Continued) 2005, provisional application No. 60/771,900, filed on Feb. 10, 2006, provisional application No. 60/814,522, OTHER PUBLICATIONS filed on Jun. 19, 2006. Rudikoffetal PNAS, 79:1979-1983, 1982.* (51) Int. Cl. A 6LX39/395 (2006.01) (Continued) A6 IK39/00 (2006.01) (52) stian (2006.01) Primary Examiner — Daniel E. Kolker USPC ...... 424/1301; 424/133.1; 424/1341; Assistant Examiner Aditi Dutt 424/135.1; 424/570 (74) Attorney, Agent, or Firm — Sterne, Kessler, Goldstein (58) Field of Classification Search & Fox PL.L.C. None See application file for complete search history. (57) ABSTRACT (56) References Cited Endogenous Sp35 is a negative regulator for neuronal Sur U.S. PATENT DOCUMENTS vival, axon regeneration, oligodendrocyte differentiation and myelination (Negative Regulator). Molecules that block 4,399.216 A 8, 1983 Axel et al. 4,444,887 A 4, 1984 Hoffman endogenous Sp35 function, such anti-Sp35 antibodies can be 4,510,245 A 4, 1985 Cousens et al. used as therapeutics for the treatment of neuron and oligo 4,634,665 A 1, 1987 Axel et al. dendrocyte dysfunction. The present invention provides anti 4,694,778 A 9, 1987 Learnet al. 4,716,111 A 12/1987 Osband et al. bodies specific for Sp35, and methods of using such antibod 4,816,397 A 3, 1989 BOSS et al. ies as antagonists of endogenous Sp35 function. The 4,816,567 A 3/1989 Cabilly et al. invention further provides specific hybridoma and phage 4.946,778 A 8, 1990 Ladner et al. library-derived monoclonal antibodies, nucleic acids encod 4,968,615 A 11, 1990 Koszinowski et al. 5,122,464 A 6, 1992 Wilson et al. ing these antibodies, and vectors and host cells comprising 5,168,062 A 12/1992 Stinski these antibodies. The invention further provides methods of 5,179,017 A 1/1993 Axel et al. promoting oligodendrocyte Survival and myelination in a ver 5,223,409 A 6, 1993 Ladner et al. tebrate, comprising administering to a vertebrate in need of 5,225,539 A 7, 1993 Winter 5,252,714 A 10, 1993 Harris et al. such treatment an effective amount of an anti-Sp35 antibody. 5,258.498 A 11/1993 Huston et al. 5,314.995 A 5/1994 Fell, Jr. et al. 5,403.484 A 4/1995 Ladner et al. 16 Claims, 11 Drawing Sheets US 8,551,476 B2 Page 2

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(56) References Cited NCBI Entrez, Accession No. DR000281, (first available May 17, 2005; last updated May 17, 2005). OTHER PUBLICATIONS NCBI Entrez, Accession No. AY324320, (first available May 4, 2004; last updated May 4, 2004). Wang, K.C., et al., “Oligodendrocyte-Myelin Glycoprotein is a Nogo NCBI Entrez, Accession No. AY324322, (first available May 4, 2004; Receptor Ligand That Inhibits Neurite Outgrowth.” Nature 417:941 last updated May 4, 2004). 944, Nature Publishing Group (2002). NCBI Entrez, Accession No. AY324323, (first available May 4, 2004; last updated May 4, 2004). Williams, E.-J. and Doherty, P., “Evidence for and against a Pivotal U.S. Appl. No. 12.092,662, inventors Mi et al., National Phase of Role of PI3-Kinase in a Neuronal Cell Survival Pathway.” Molec. PCT/US2006/042990, international filing date of Nov. 3, 2006 (Not Cell. Neurosci. 13:272-280, Academic Press (1999). Published). Xu, W., et al., “Chaperone-dependent E3 ubiquitin ligase CHIP medi U.S. Appl. No. 12/095,857, inventors Mi et al., National Phase of ates a degradative pathway for c-ErbB2/Neu.” Proc. Natl. Acad. Sci. PCT/US2006/045993, international filing date of Dec. 1, 2006 (Not USA 99:12847-12852, National Academy of Sciences (2002). Published). Yang, L., et al., “A novel azulenyl nitrone antioxidant protects against Office Action mailed Oct. 1, 2012, in Japanese Patent Application MPTP and 3-nitropropionic acid neurotoxicities.” Exp. Neurol. No. 2008-520375 (English language translation included). 191:86-93, Elsevier Inc. (Jan. 2005). Paul, WE, ed. Fundamental Immunology. Third Edition. Raven Yu, W., et al., “Segregation of Nogo66 receptors into lipid rafts in rat Press, New York, 1993, pp. 292-295, under the heading “Fv Structure brain and inhibition of Nogo66 signaling by cholesterol depletion.” and Diversity in Three Dimensions.” FEBS Lett. 577:87-92, Elsevier B.V. (Nov. 2004). U.S. Appl. No. 13/414.222, inventors Mi et al., filed Mar. 7, 2012. Zhou, P. et al., “ErbB2 Degradation Mediated by the Co-chaperone U.S. Appl. No. 13/356,413, inventors Mi et al., filed Jan. 23, 2012. Protein CHIP.J. Biol. Chem. 278: 13829-13837. The American Soci Office Action mailed Jun. 24, 2011, in U.S. Appl. No. 12.092,662, ety for Biochemistry and Molecular Biology, Inc. (2003). Mi, filed Sep. 26, 2008. NCBI Entrez. Accession No. BC011057, (first available Jul. 30, Office Action mailed Feb. 9, 2012, in U.S. Appl. No. 12/092,662, Mi, 2001; last updated Feb. 8, 2007). filed Sep. 26, 2008. NCBI Entrez, Accession No. BC068558, (first available Apr. 6, 2004; Office Action mailed Oct. 25, 2011, in U.S. Appl. No. 13/158,307. last updated Feb. 8, 2007). Mi, filed Jun. 10, 2011. NCBI Entrez, Accession No. NM 152570, (first available Sep. 6, Office Action mailed, Aug. 20, 2012 in U.S. Appl. No. 13/158.307. 2002; last updated Feb. 11, 2008). Office Action mailed, Jul. 24, 2012 in U.S. Appl. No. 1 1/165,576. NCBI Entrez, Accession No. NM 032808, (first available May 31, 2001; last updated Feb. 11, 2008). * cited by examiner U.S. Patent Oct. 8, 2013 Sheet 1 of 11 US 8,551,476 B2

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Lo C lo C 3 N O CN O (ue'SESuoxe) uObelieuefie US 8,551,476 B2 1. 2 SP35 ANTIBODIES AND USES THEREOF proteins (e.g., NogoA, MAG and OM-gp), the NgR1 complex transduces signals that lead to growth cone collapse and inhi CROSS REFERENCE TO RELATED bition of neurite outgrowth. APPLICATIONS There is an unmet need for molecules and methods for inhibiting NgR1-mediated growth cone collapse and the The present application is a continuation of International resulting inhibition of neurite outgrowth. Additionally there Application No. PCT/US2006/026271, filed Jul. 7, 2006, is a need for molecules which increase neuronal Survival and which claims the benefit of U.S. Provisional Application No. axon regeneration. Particularly for the treatment of disease, 60/814.522, filed Jun. 19, 2006, U.S. Provisional Application disorders or injuries which involve axonal injury, neuronal or 10 oligodendrocyte cell death, demyelination or dymyelination No. 60/771,900, filed Feb. 10, 2006 and U.S. Provisional or generally relate to the nervous system. Application No. 60/697.336, filed Jul. 8, 2005, all of which Such diseases, disorders or injuries include, but are not are incorporated herein by reference in their entireties. limited to, multiple sclerosis (MS), progressive multifocal BACKGROUND OF THE INVENTION leukoencephalopathy (PML), encephalomyelitis (EPL), cen 15 tral pontine myelolysis (CPM), adrenoleukodystrophy, Alex ander's disease, Pelizaeus Merzbacher disease (PMZ), 1. Field of the Invention Globoid cell Leucodystrophy (Krabbe's disease) and Walle This invention relates to neurology, neurobiology and rian Degeneration, optic neuritis, transverse myelitis, amy molecular biology. More particularly, this invention relates to lotrophic lateral Sclerosis (ALS), Huntington's disease, molecules and methods for treatment of neurological dis Alzheimer's disease, Parkinson's disease, spinal cord injury, eases, disorders and injuries such as spinal cord injury. traumatic brain injury, post radiation injury, neurologic com 2. Background of the Invention plications of chemotherapy, stroke, acute ischemic optic neu Axons and dendrites extend from neurons. The distal tip of ropathy, vitamin E deficiency, isolated vitamin E deficiency an extending axon or neurite includes a specialized region, syndrome, AR, Bassen-KornZweig syndrome, Marchiafava known as the growth cone. Growth cones sense the local 25 Bignami syndrome, metachromatic leukodystrophy, trigemi environment and guide axonal growth toward a neuron's tar nal neuralgia, and Bell's palsy. Among these diseases, MS is get cell. Growth cones respond to environmental cues, for the most widespread, affecting approximately 2.5 million example, Surface adhesiveness, growth factors, neurotrans people worldwide. mitters and electric fields. The growth cones generally MS generally begins with a relapsing-remitting pattern of advance at a rate of one to two millimeters per day. The 30 neurologic involvement, which then progresses to a chronic growth cone explores the area ahead of it and on either side, phase with increasing neurological damage. MS is associated by means of elongations classified as lamellipodia and filopo with the destruction of myelin, oligodendrocytes and axons dia. When an elongation contacts an unfavorable Surface, it localized to chronic lesions. The demyelination observed in withdraws. When an elongation contacts a favorable growth MS is not always permanent and remyelination has been documented in early stages of the disease. Remyelination of Surface, it continues to extend and guides the growth cone in neurons requires oligodendrocytes. that direction. When the growth cone reaches an appropriate Various disease-modifying treatments are available for target cell a synaptic connection is created. MS, including the use of corticosteroids and immunomodu Nerve cell function is influenced by contact between neu lators such as interferon beta and Tysabri R. In addition, rons and other cells in their immediate environment (Rut 40 because of the central role of oligodendrocytes and myelina ishauser, et al., 1988, Physiol. Rev. 68:819). These cells tion in MS, there have been efforts to develop therapies to include specialized glial cells, oligodendrocytes in the central increase oligodendrocyte numbers or enhance myelination. nervous system (CNS), and Schwann cells in the peripheral See, e.g., Cohen et al., U.S. Pat. No. 5,574,009; Chang et al., nervous system (PNS), which sheathe the neuronal axon with N. Engl. J. Med. 346: 165-73 (2002). However, there remains myelin (Lemke, 1992, in An Introduction to Molecular Neu 45 an urgent need to devise additional therapies for MS and other robiology, Z. Hall, Ed., p. 281, Sinauer). demyelination and dismyelination disorders. CNS neurons have the inherent potential to regenerate after injury, but they are inhibited from doing so by inhibitory BRIEF SUMMARY OF THE INVENTION proteins present in myelin (Brittis et al., 2001, Neuron 30:11 14; Jones et al., 2002, J. Neurosci. 22:2792-2803; Grimpe et 50 The present invention is based on the discovery that Sp35 al., 2002, J. Neurosci. 22:3144-3160). (Sp35 is also designated in the literature as LINGO-1 and Several myelin inhibitory proteins found on oligodendro LRRN6) is expressed in oligodendrocytes and neuronal cells cytes have been characterized. Known examples of myelin and negatively regulates oligodendrocyte/neuronal differen inhibitory proteins include Nogo-A (Chen et al., Nature, 2000, tiation, Survival and axon myelination. Furthermore, certain 403, 434-439; Grandpre et al., Nature 2000, 403, 439-444), 55 antagonists of Sp35 promote survival, proliferation and dif myelin associated glycoprotein (MAG) (McKerracher et al., ferentiation of oligodendrocytes and neuronal cells, as well as 1994, Neuron 13:805-811; Mukhopadhyay et al., 1994, Neu myelination of neurons. Based on these discoveries, the rom 13:757-767) and oligodendrocyte glycoprotein (OM-gp), invention relates generally to antibodies, antigen binding Mikol et al., 1988, J. Cell. Biol. 106:1273-1279). Each of fragment or derivatives thereof which can be used as an these proteins has been separately shown to be a ligand for the 60 antagonist of Sp35. Additionally, the invention generally neuronal Nogo receptor-1 (NgR1 (Wang et al., Nature 2002, relates to methods for treating various disease, disorders or 417. 941-944; Grandpre et al., Nature 2000, 403, 439-444; injuries associated with demyelination, dysmyelination, oli Chen et al., Nature, 2000, 403, 434-439; Domeniconi et al., godendrocyte/neuronal cell death or axonal injury by the Neuron 2002, published online Jun. 28, 2002). administration of an Sp35 antagonist antibody or antigen Nogo receptor-1 (NgR1) is a GPI-anchored membrane 65 binding fragment. protein that contains 8 leucine rich repeats (Fournier et al., In certain embodiments, the invention includes an isolated 2001, Nature 409:341-346). Upon interaction with inhibitory antibody or antigen binding fragment thereof which specifi US 8,551,476 B2 3 4 cally binds to the same Sp35 epitope as a reference mono In certain embodiments, the invention includes composi clonal antibody selected from the group consisting of 201'. tions comprising the antibodies or antigenbinding fragments 3A3, 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3, described herein. 3P1E1 1.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, In additional embodiments, the invention includes meth 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1 D8, 3P4C8.2G9, 30-C12 ods for treating CNS injury, ALS, Huntington's disease, (LiO1), 38-D01 (LiO2), 35-E04 (LiO3), 36-C09 (LiO4), Alzheimer's disease, Parkinson's disease, diabetic neuropa 30-A11 (LiO5), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 thy and stroke comprising administering to an animal in need (LiO8), 36-A12 (LiO9), 28-D02 (Li10), 30-B01 (Li11), of said treatment an effective amount of an agent selected 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495 from the group consisting of an isolated Sp35 antibody or (L1 a.2), 3563 (L1 a.3), 3564 (L1 a.4), 3565 (L1a.5), 3566 10 fragment thereof or compositions comprising said antibody (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 or fragment thereof. (L1a. 10), 3571 (L1a. 11), 3582 (L1a.12), and 1968 (L1a.13). In other embodiments, the invention includes methods for Certain embodiments of the invention include an isolated treating disease or disorders associated with inhibition of polypeptide comprising an immunoglobulin heavy chain oligodendrocyte growth or differentiation; demyelination or 15 dysmyelination of CNS neurons including multiple sclerosis variable region (VH) wherein the CDR1, CDR2 and CDR3 (MS), progressive multifocal leukoencephalopathy (PML), regions are selected from the polypeptide sequences shown in encephalomyelitis (EPL), central pontine myelolysis (CPM), Table 4 or at least 80%, 85%, 90 or 95% identical to the Wallerian Degeneration, adrenoleukodystrophy, Alexanders polypeptide sequences shown in Table 4. disease, and Pelizaeus Merzbacher disease (PMZ) by admin Certain embodiments of the invention include an isolated istering to an animal in need of said treatment an effective polypeptide comprising an immunoglobulin light chain vari amount of an agent selected from the group consisting of an able region (VL) wherein the CDR1, CDR2 and CDR3 isolated Sp35 antibody or fragment thereof or compositions regions are selected from the polypeptide sequences shown in comprising said antibody or fragment thereof. Table 5 or at least 80%, 85%, 90% or 95% identical to the Other embodiments of the present invention include a polypeptide sequences shown in Table 5. 25 method of inhibiting signal transduction by Nogo receptor 1 Certain embodiments of the invention include an isolated (NgR1), comprising contacting the NgR1 with an effective polypeptide comprising an immunoglobulin heavy chain amount of an agent selected from the group consisting of the variable region (VH) selected from the group consisting of isolated Sp35 antibody or fragment thereof or compositions SEQID NOs: 158 to 172,372,376,380 and 384, as shown in comprising said antibody or fragment thereof. Table 6, or at least 80%, 85%, 90% or 95% identical to said 30 Additional embodiments of the present invention include a SEQID NOs: 158 to 172,372,376,380 and 384 as shown in method of decreasing inhibition of axonal growth of a central Table 6. nervous system (CNS) neuron, comprising contacting the Certain embodiments of the invention include an isolated neuron with an effective amount of an agent selected from the polypeptide comprising an immunoglobulin light chain vari group consisting of the isolated Sp35 antibody or fragment 35 thereof of or compositions comprising said antibody or frag able region (VL) selected from the group consisting of SEQ ment thereof. ID NOS: 273 to 286,373,377,381 and 385, as shown in Table Other embodiments of the present invention include a 8, or at least 80%, 85%, 90% or 95% identical to said SEQID method of inhibiting growth cone collapse of a CNS neuron, NOS: 273 to 286, 373,377,381 and 385, as shown in Table 8. comprising contacting the neuron with an effective amount of In additional embodiments, the invention includes an iso 40 an agent selected from the group consisting of the isolated lated polynucleotide comprising a nucleic acid encoding an Sp35 antibody or fragment thereof or compositions compris immunoglobulin heavy chain variable region (VH) wherein ing said antibody or fragment thereof. the CDR1, CDR2 and CDR3 regions are selected from the group selected from the polynucleotide sequences shown in BRIEF DESCRIPTION OF THE Table 4 or at least 80%, 85%, 90 or 95% identical to the 45 DRAWINGS/FIGURES polynucleotide sequences shown in Table 4. In other embodiments, the invention includes an isolated FIG. 1: SDS-PAGE gel showing immunoprecipitation of polynucleotide comprising a nucleic acid encoding an immu Sp35 by monoclonal antibodies 1A7 and 2F3. noglobulin light chain variable region (VL) wherein the FIG. 2: FACS result showing that MAbs 1A7 and 2F3 CDR1, CDR2 and CDR3 regions are selected from the poly 50 bound to COS-7 or 293 cells expressing Sp35, but not to nucleotide sequences shown in Table 5 or at least 80%, 85%, control cells with no Sp35 expression. 90% or 95% identical to the polynucleotide sequences shown FIG. 3: MAbs 1A7 and 2F3 protected DRG neurons from in Table 5. myelin-mediated inhibition of neurite outgrowth. Other embodiments of the invention include, an isolated FIG. 4A-G: Immunohistochemical staining (“IHC) of polynucleotide comprising a nucleic acid encoding an immu 55 cocultures of DRG neurons and oligodendrocytes treated noglobulin heavy chain variable region (VH) selected from with monoclonal antibodies 1A7 and 2F3, or control anti the group consisting of SEQID NOs: 173 to 184, 370, 374, body. Panels D and E are enlargements of panels B and C, 378 and 382, as shown in Table 7, or at least 80%, 85%, 90% respectively. Staining with anti-fIII-tubulin antibody to iden or 95% identical to said SEQID NOs: 173 to 184,370,374, tify axons, or anti-MBP antibody to identify oligodendro 378 and 382, as shown in Table 7. 60 cytes. F: Quantitation of MBP+ myelinating cells upon treat Other embodiments of the invention include, an isolated ment of cocultures with 1A7 or 2F3. G. Western blot analysis polynucleotide comprising a nucleic acid encoding an immu to quantify the MBP produced from cocultures of DRG neu noglobulin light chain variable region (VL) selected from the rons and oligodendrocytes treated with monoclonal antibod group consisting of SEQID NOs: 185 to 194,371,375, 379 ies 1A7 and 2F3. and 383, as shown in Table 9, or at least 80%, 85%, 90% or 65 FIG. 5A-C: A: CC1 antibody staining of mouse oligoden 95% identical to said SEQID NOs: 185 to 194,371,375,379 drocytes in cuprizone model. B. Anti-MBP protein antibody and 383, as shown in Table 9. or luxol fast blue staining of mouse neurons in cuprizone US 8,551,476 B2 5 6 model. C. Quantitation of CC1 antibody-positive oligoden protein coupled to at least one carbohydrate moiety that is drocytes at four weeks and 6 weeks. attached to the protein via an oxygen-containing or a nitro FIG. 6: Surviving RGCs. Treatment with monoclonal anti gen-containing side chain of an amino acid residue, e.g., a body 1A7 Anti-Sp35 antibody 1A7 treated animals showed serine residue or an asparagine residue. significant neuronal survival (80%) when compared to con- 5 By an "isolated polypeptide or a fragment, variant, or trol-antibody or PBS treated animals, which each only derivative thereof is intended a polypeptide that is not in its showed approximately 50% neuronal survival. natural milieu. No particular level of purification is required. FIG. 7. BBB scores of mice receiving anti-Sp35 antibody For example, an isolated polypeptide can be removed from its 1A7 after spinal cord injury as described in Example 8. native or natural environment. Recombinantly produced FIG.8. Western blot of co-cultured oligodendrocytes and 10 polypeptides and proteins expressed in host cells are consid DRGs after incubation with anti-Sp35 antibodies Li05, Li06 ered isolated for purposed of the invention, as are native or and 3, 10 and 30 mg of Sp35-Fc (LINGO-1-Ig) as described recombinant polypeptides which have been separated, frac in Example 9. tionated, or partially or substantially purified by any suitable FIG.9. Photographs of the optic nerves of A) Normal Rats: technique. B) Myelin Oligodendrocyte Glycoprotein (MOG) induced 15 Also included as polypeptides of the present invention are Experimental Autoimmune Encephalomyelitis (EAE) rats: fragments, derivatives, analogs, or variants of the foregoing and C) Myelin Oligodendrocyte Glycoprotein (MOG) polypeptides, and any combination thereof. The terms “frag induced Experimental Autoimmune Encephalomyelitis ment.” “variant.” “derivative' and “analog when referring to (EAE) rats treated with the Sp35 antibody 1A7. Electron Sp35 antibodies or antibody polypeptides of the present micrographs of each optic nerve are shown below each pho- 20 invention include any polypeptides which retain at least some tograph of the optic nerve. of the antigen-binding properties of the corresponding native FIG. 10. Graph of the number of regenerative neuronal antibody or polypeptide. Fragments of polypeptides of the fibers per section counted in animals receiving an intravitreal present invention include proteolytic fragments, as well as injection of the Sp35 antibody 1A7 after optic nerve crush. deletion fragments, in addition to specificantibody fragments 25 discussed elsewhere herein. Variants of Sp35 antibodies and DETAILED DESCRIPTION OF THE INVENTION antibody polypeptides of the present invention include frag ments as described above, and also polypeptides with altered I. Definitions amino acid sequences due to amino acid Substitutions, dele tions, or insertions. Variants may occur naturally or be non It is to be noted that the term “a” or “an entity refers to one 30 naturally occurring Non-naturally occurring variants may be or more of that entity; for example, “an Sp35 antibody, is produced using art-known mutagenesis techniques. Variant understood to represent one or more Sp35 antibodies. As polypeptides may comprise conservative or non-conservative such, the terms “a” (or “an”), “one or more.” and “at least one' amino acid substitutions, deletions or additions. Derivatives can be used interchangeably herein. of Sp35 antibodies and antibody polypeptides of the present As used herein, the term “polypeptide' is intended to 35 invention, are polypeptides which have been altered so as to encompass a singular “polypeptide' as well as plural exhibit additional features not found on the native polypep "polypeptides, and refers to a molecule composed of mono tide. Examples include fusion proteins. Variant polypeptides mers (amino acids) linearly linked by amide bonds (also may also be referred to herein as “polypeptide analogs. As known as peptide bonds). The term “polypeptide' refers to used herein a "derivative' of an Sp35 antibody or antibody any chain or chains of two or more amino acids, and does not 40 polypeptide refers to a subject polypeptide having one or refer to a specific length of the product. Thus, peptides, dipep more residues chemically derivatized by reaction of a func tides, tripeptides, oligopeptides, “protein.” “amino acid tional side group. Also included as "derivatives” are those chain.” or any other term used to refer to a chain or chains of peptides which contain one or more naturally occurring two or more amino acids, are included within the definition of amino acid derivatives of the twenty standard amino acids. "polypeptide.” and the term “polypeptide' may be used 45 For example, 4-hydroxyproline may be substituted for pro instead of, or interchangeably with any of these terms. The line:5-hydroxylysine may be substituted for lysine; 3-meth term “polypeptide' is also intended to refer to the products of ylhistidine may be substituted for histidine; homoserine may post-expression modifications of the polypeptide, including be substituted for serine; and ornithine may be substituted for without limitation glycosylation, acetylation, phosphoryla lysine. tion, amidation, derivatization by known protecting/blocking 50 The term “polynucleotide' is intended to encompass a groups, proteolytic cleavage, or modification by non-natu singular nucleic acid as well as plural nucleic acids, and refers rally occurring amino acids. A polypeptide may be derived to an isolated nucleic acid molecule or construct, e.g., mes from a natural biological source or produced by recombinant senger RNA (mRNA) or plasmid DNA (pDNA). A poly technology, but is not necessarily translated from a desig nucleotide may comprise a conventional phosphodiester nated nucleic acid sequence. It may be generated in any 55 bond or a non-conventional bond (e.g., an amide bond. Such manner, including by chemical synthesis. as found in peptide nucleic acids (PNA)). The term “nucleic A polypeptide of the invention may be of a size of about 3 acid refer to any one or more nucleic acid segments, e.g., or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or DNA or RNA fragments, present in a polynucleotide. By more, 75 or more, 100 or more, 200 or more, 500 or more, "isolated nucleic acid or polynucleotide is intended a nucleic 1,000 or more, or 2,000 or more amino acids. Polypeptides 60 acid molecule, DNA or RNA, which has been removed from may have a defined three-dimensional structure, although its native environment. For example, a recombinant poly they do not necessarily have such structure. Polypeptides nucleotide encoding an Sp35 antibody contained in a vectoris with a defined three-dimensional structure are referred to as considered isolated for the purposes of the present invention. folded, and polypeptides which do not possess a defined Further examples of an isolated polynucleotide include three-dimensional structure, but rather can adopt a large num- 65 recombinant polynucleotides maintained in heterologous ber of different conformations, and are referred to as host cells or purified (partially or substantially) polynucle unfolded. As used herein, the term glycoprotein refers to a otides in solution. Isolated RNA molecules include in vivo or US 8,551,476 B2 7 8 in vitro RNA transcripts of polynucleotides of the present eukaryotic cells. Additional Suitable transcription control invention. Isolated polynucleotides or nucleic acids accord regions include tissue-specific promoters and enhancers as ing to the present invention further include Such molecules well as lymphokine-inducible promoters (e.g., promoters produced synthetically. In addition, polynucleotide or a inducible by interferons or interleukins). nucleic acid may be or may include a regulatory element Such 5 Similarly, a variety of translation control elements are as a promoter, ribosome binding site, or a transcription ter known to those of ordinary skill in the art. These include, but minator. are not limited to ribosome binding sites, translation initiation As used herein, a “coding region' is a portion of nucleic and termination codons, and elements derived from picor acid which consists of codons translated into amino acids. naviruses (particularly an internal ribosome entry site, or Although a “stop codon” (TAG, TGA, or TAA) is not trans- 10 IRES, also referred to as a CITE sequence). lated into an amino acid, it may be considered to be part of a In other embodiments, a polynucleotide of the present coding region, but any flanking sequences, for example pro invention is RNA, for example, in the form of messenger moters, ribosome binding sites, transcriptional terminators, RNA (mRNA). introns, and the like, are not part of a coding region. Two or Polynucleotide and nucleic acid coding regions of the more coding regions of the present invention can be present in 15 present invention may be associated with additional coding a single polynucleotide construct, e.g., on a single vector, or regions which encode secretory or signal peptides, which in separate polynucleotide constructs, e.g., on separate (dif direct the secretion of a polypeptide encoded by a polynucle ferent) vectors. Furthermore, any vector may contain a single otide of the present invention. According to the signal hypoth coding region, or may comprise two or more coding regions, esis, proteins secreted by mammalian cells have a signal e.g., a single vector may separately encode an immunoglobu- 20 peptide or secretory leader sequence which is cleaved from lin heavy chain variable region and an immunoglobulin light the mature protein once export of the growing protein chain chain variable region. In addition, a vector, polynucleotide, or across the rough endoplasmic reticulum has been initiated. nucleic acid of the invention may encode heterologous coding Those of ordinary skill in the art are aware that polypeptides regions, either fused or unfused to a nucleic acid encoding an secreted by Vertebrate cells generally have a signal peptide Sp35 antibody or fragment, variant, or derivative thereof. 25 fused to the N-terminus of the polypeptide, which is cleaved Heterologous coding regions include without limitation spe from the complete or “full length” polypeptide to produce a cialized elements or motifs, such as a secretory signal peptide secreted or “mature' form of the polypeptide. In certain or a heterologous functional domain. embodiments, the native signal peptide, e.g., an immunoglo In certain embodiments, the polynucleotide or nucleic acid bulin heavy chain or light chain signal peptide is used, or a is DNA. In the case of DNA, a polynucleotide comprising a 30 functional derivative of that sequence that retains the ability nucleic acid which encodes a polypeptide normally may to direct the secretion of the polypeptide that is operably include a promoter and/or other transcription or translation associated with it. Alternatively, a heterologous mammalian control elements operably associated with one or more cod signal peptide, or a functional derivative thereof, may be used. ing regions. An operable association is when a coding region For example, the wild-type leader sequence may be substi for a gene product, e.g., a polypeptide, is associated with one 35 tuted with the leader sequence of human tissue plasminogen or more regulatory sequences in Such a way as to place activator (TPA) or mouse B-glucuronidase. expression of the gene product under the influence or control The present invention is directed to certain Sp35 antibod of the regulatory sequence(s). Two DNA fragments (such as a ies, or antigen-binding fragments, variants, or derivatives polypeptide coding region and a promoter associated there thereof. Unless specifically referring to full-sized antibodies with) are “operably associated if induction of promoter 40 such as naturally-occurring antibodies, the term “Sp35 anti function results in the transcription of mRNA encoding the bodies' encompasses full-sized antibodies as well as antigen desired gene product and if the nature of the linkage between binding fragments, variants, analogs, or derivatives of Such the two DNA fragments does not interfere with the ability of antibodies, e.g., naturally occurring antibody or immunoglo the expression regulatory sequences to direct the expression bulin molecules or engineered antibody molecules or frag of the gene product or interfere with the ability of the DNA 45 ments that bind antigen in a manner similar to antibody mol template to be transcribed. Thus, a promoter region would be ecules. operably associated with a nucleic acid encoding a polypep The terms “antibody” and “immunoglobulin' are used tide if the promoter was capable of effecting transcription of interchangeably herein. An antibody or immunoglobulin that nucleic acid. The promoter may be a cell-specific pro comprises at least the variable domain of a heavy chain, and moter that directs substantial transcription of the DNA only in 50 normally comprises at least the variable domains of a heavy predetermined cells. Other transcription control elements, chain and a light chain. Basic immunoglobulin structures in besides a promoter, for example enhancers, operators, repres vertebrate systems are relatively well understood. See, e.g., sors, and transcription termination signals, can be operably Harlow et al., Antibodies: A Laboratory Manual, (Cold associated with the polynucleotide to direct cell-specific tran Spring Harbor Laboratory Press, 2nd ed. 1988). scription. Suitable promoters and other transcription control 55 As will be discussed in more detail below, the term “immu regions are disclosed herein. noglobulin' comprises various broad classes of polypeptides A variety of transcription control regions are known to that can be distinguished biochemically. Those skilled in the those skilled in the art. These include, without limitation, art will appreciate that heavy chains are classified as gamma, transcription control regions which function in Vertebrate mu, alpha, delta, or epsilon, (Y, L, C, 6, e) with some Sub cells, such as, but not limited to, promoter and enhancer 60 classes among them (e.g., Y1-y4). It is the nature of this chain segments from cytomegaloviruses (the immediate early pro that determines the “class of the antibody as IgG, IgM, IgA moter, in conjunction with intron-A), simian virus 40 (the IgG, or IgE, respectively. The immunoglobulin Subclasses early promoter), and retroviruses (such as Rous sarcoma (isotypes) e.g., IgG, IgG, IgG, IgG, IgA, etc. are well virus). Other transcription control regions include those characterized and are known to confer functional specializa derived from vertebrate genes such as actin, heat shock pro- 65 tion. Modified versions of each of these classes and isotypes tein, bovine growth hormone and rabbit B-globin, as well as are readily discernable to the skilled artisan in view of the other sequences capable of controlling gene expression in instant disclosure and, accordingly, are within the scope of the US 8,551,476 B2 10 instant invention. All immunoglobulin classes are clearly Surface promotes the non-covalent binding of the antibody to within the scope of the present invention, the following dis its cognate epitope. The amino acids comprising the CDRS cussion will generally be directed to the IgG class of immu and the framework regions, respectively, can be readily iden noglobulin molecules. With regard to IgG, a standard immu tified for any given heavy or light chain variable region by one noglobulin molecule comprises two identical light chain of ordinary skill in the art, since they have been precisely polypeptides of molecular weight approximately 23,000 Dal defined (see, “Sequences of Proteins of Immunological Inter tons, and two identical heavy chain polypeptides of molecular est.” Kabat, E., et al., U.S. Department of Health and Human weight 53,000-70,000. The four chains are typically joined by Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196: disulfide bonds in a “Y” configuration wherein the light 901-917 (1987), which are incorporated herein by reference chains bracket the heavy chains starting at the mouth of the 10 “Y” and continuing through the variable region. in their entireties). Light chains are classified as either kappa or lambda (K, w). In the case where there are two or more definitions of a term Each heavy chain class may be bound with either a kappa or which is used and/or accepted within the art, the definition of lambda light chain. In general, the light and heavy chains are the term as used herein is intended to include all such mean covalently bonded to each other, and the “tail portions of the 15 ings unless explicitly stated to the contrary. A specific two heavy chains are bonded to each other by covalent disul example is the use of the term “complementarity determining fide linkages or non-covalent linkages when the immunoglo region” (“CDR) to describe the non-contiguous antigen bulins are generated either by hybridomas, B cells or geneti combining sites found within the variable region of both cally engineered host cells. In the heavy chain, the amino acid heavy and light chain polypeptides. This particular region has sequences run from an N-terminus at the forked ends of the Y been described by Kabat et al., U.S. Dept. of Health and configuration to the C-terminus at the bottom of each chain. Human Services, “Sequences of Proteins of Immunological Both the light and heavy chains are divided into regions of Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901 structural and functional homology. The terms "constant and 917 (1987), which are incorporated herein by reference, “variable' are used functionally. In this regard, it will be where the definitions include overlapping or Subsets of amino appreciated that the variable domains of both the light (V) 25 acid residues when compared against each other. Neverthe and heavy (V) chain portions determine antigen recognition less, application of either definition to refer to a CDR of an and specificity. Conversely, the constant domains of the light antibody or variants thereof is intended to be within the scope chain (C) and the heavy chain (C1, C2 or C3) confer of the term as defined and used herein. The appropriate amino important biological properties such as secretion, transpla acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as cental mobility, Fc receptor binding, complement binding, 30 and the like. By convention the numbering of the constant a comparison. The exact residue numbers which encompass a region domains increases as they become more distal from the particular CDR will vary depending on the sequence and size antigen binding site or amino-terminus of the antibody. The of the CDR. Those skilled in the art can routinely determine N-terminal portion is a variable region and at the C-terminal which residues comprise a particular CDR given the variable portion is a constant region; the C3 and C, domains actually 35 region amino acid sequence of the antibody. comprise the carboxy-terminus of the heavy and light chain, respectively. TABLE 1 As indicated above, the variable regionallows the antibody CDR Definitions' to selectively recognize and specifically bind epitopes on antigens. That is, the V, domain and V. domain, or subset of 40 Kabat Chothia the complementarity determining regions (CDRS), of an anti V. CDR1 31-35 26-32 body combine to form the variable region that defines a three V. CDR2 SO-65 52-58 dimensional antigen binding site. This quaternary antibody V. CDR3 95-102 95-102 structure forms the antigen binding site present at the end of V, CDR1 24-34 26-32 V, CDR2 SO-56 50-52 each arm of the Y. More specifically, the antigen binding site 45 is defined by three CDRs on each of the V and V, chains. In V, CDR3 89-97 91-96 Some instances, e.g., certain immunoglobulin molecules "Numbering of all CDR definitions in Table 1 is according to the numbering conventions set derived from camelid species or engineered based on camelid forth by Kabat et al. (see below). immunoglobulins, a complete immunoglobulin molecule Kabat et al. also defined a numbering system for variable may consist of heavy chains only, with no light chains. See, 50 domain sequences that is applicable to any antibody. One of e.g., Hamers-Casterman et al., Nature 363:446-448 (1993). ordinary skill in the art can unambiguously assign this system In naturally occurring antibodies, the six "complementar of "Kabat numbering to any variable domain sequence, ity determining regions' or “CDRs' present in each antigen without reliance on any experimental data beyond the binding domain are short, non-contiguous sequences of sequence itself. As used herein, “Kabat numbering refers to amino acids that are specifically positioned to form the anti 55 the numbering system set forth by Kabat et al., U.S. Dept. of gen binding domain as the antibody assumes its three dimen Health and Human Services, “Sequence of Proteins of Immu sional configuration in an aqueous environment. The remain nological Interest' (1983). Unless otherwise specified, refer der of the amino acids in the antigen binding domains, ences to the numbering of specific amino acid residue posi referred to as “framework” regions, show less inter-molecular tions in an Sp35 antibody or antigen-binding fragment, variability. The framework regions largely adopt a B-sheet 60 variant, or derivative thereof of the present invention are conformation and the CDRs form loops which connect, and in according to the Kabat numbering system. Some cases form part of the B-sheet structure. Thus, frame In camelid species, the heavy chain variable region, work regions act to form a scaffold that provides for position referred to as V.H., forms the entire antigen-binding domain. ing the CDRS in correctorientation by inter-chain, non-cova The main differences between camelid VH variable regions lent interactions. The antigen binding domain formed by the 65 and those derived from conventional antibodies (V) include positioned CDRs defines a surface complementary to the (a) more hydrophobic amino acids in the light chain contact epitope on the immunoreactive antigen. This complementary Surface of V as compared to the corresponding region in US 8,551,476 B2 11 12 V.H., (b) a longer CDR3 in VH, and (c) the frequent occur monomer may comprise a different target binding site, form rence of a disulfide bond between CDR1 and CDR3 in V.H. ing, for example, a bispecific antibody. Antibodies or antigen-binding fragments, variants, or The heavy chain portions of a binding polypeptide for use derivatives thereof of the invention include, but are not lim in the diagnostic and treatment methods disclosed herein may ited to, polyclonal, monoclonal, multispecific, human, 5 be derived from different immunoglobulin molecules. For humanized, primatized, or chimeric antibodies, single chain example, a heavy chain portion of a polypeptide may com antibodies, epitope-binding fragments, e.g., Fab., Fab' and prise a C1 domain derived from an IgG1 molecule and a F(ab'), Fd, FVs, single-chain FVS (ScPV), single-chain anti hinge region derived from an IgG3 molecule. In another bodies, disulfide-linked FVS (SdPV), fragments comprising example, a heavy chain portion can comprise a hinge region either a V, or V. domain, fragments produced by a Fab 10 expression library, and anti-idiotypic (anti-Id) antibodies (in derived, in part, from an IgG1 molecule and, in part, from an cluding, e.g., anti-Id antibodies to Sp35 antibodies disclosed IgG3 molecule. In another example, a heavy chain portion herein). Schv molecules are known in the art and are can comprise a chimeric hinge derived, in part, from an IgG1 described, e.g., in U.S. Pat. No. 5,892.019. Immunoglobulin molecule and, in part, from an IgG4 molecule. or antibody molecules of the invention can be of any type 15 As used herein, the term “light chain portion' includes (e.g., IgG, IgE. IgM, Ig), IgA, and IgY), class (e.g., IgG1, amino acid sequences derived from an immunoglobulin light IgG2, IgG3, IgG4, IgA1 and IgA2) or Subclass of immuno chain. Preferably, the light chain portion comprises at least globulin molecule. one of a V, or C, domain. Antibody fragments, including single-chain antibodies, Sp35 antibodies, orantigen-binding fragments, variants, or may comprise the variable region(s) alone or in combination derivatives thereof disclosed herein may be described or with the entirety or a portion of the following: hinge region, specified interms of the epitope(s) or portion(s) of an antigen, C1, C2, and C3 domains. Also included in the invention e.g., a target polypeptide (Sp35) that they recognize or spe are antigen-binding fragments also comprising any combina cifically bind. The portion of a target polypeptide which spe tion of variable region(s) with a hinge region, C1, C2, and cifically interacts with the antigenbinding domain of an anti C3 domains. Antibodies or immunospecific fragments 25 body is an “epitope, or an “antigenic determinant.” A target thereof for use in the diagnostic and therapeutic methods polypeptide may comprise a single epitope, but typically disclosed herein may be from any animal origin including comprises at least two epitopes, and can include any number birds and mammals. Preferably, the antibodies are human, of epitopes, depending on the size, conformation, and type of murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, antigen. Furthermore, it should be noted that an “epitope' on or chicken antibodies. In another embodiment, the variable 30 a target polypeptide may be or include non-polypeptide ele region may be condricthoid in origin (e.g., from sharks). As ments, e.g., an "epitope may include a carbohydrate side used herein, "human’ antibodies include antibodies having chain. the amino acid sequence of a human immunoglobulin and The minimum size of a peptide or polypeptide epitope for include antibodies isolated from human immunoglobulin an antibody is thought to be about four to five amino acids. libraries or from animals transgenic for one or more human 35 Peptide or polypeptide epitopes preferably contain at least immunoglobulins and that do not express endogenous immu seven, more preferably at least nine and most preferably noglobulins, as described infra and, for example in, U.S. Pat. between at least about 15 to about 30 amino acids. Since a No. 5,939,598 by Kucherlapati et al. CDR can recognize an antigenic peptide or polypeptide in its As used herein, the term “heavy chain portion' includes tertiary form, the amino acids comprising an epitope need not amino acid sequences derived from an immunoglobulin 40 be contiguous, and in some cases, may not even be on the heavy chain. A polypeptide comprising a heavy chain portion same peptide chain. In the present invention, peptide or comprises at least one of a C1 domain, a hinge (e.g., upper, polypeptide epitope recognized by Sp35 antibodies of the middle, and/or lower hinge region) domain, a C2 domain, a present invention contains a sequence of at least 4, at least 5, C3 domain, or a variant or fragment thereof. For example, a at least 6, at least 7, more preferably at least 8, at least 9, at binding polypeptide for use in the invention may comprise a 45 least 10, at least 15, at least 20, at least 25, or between about polypeptide chain comprising a C1 domain; a polypeptide 15 to about 30 contiguous or non-contiguous amino acids of chain comprising a C1 domain, at least a portion of a hinge Sp35. domain, and a C2 domain; a polypeptide chain comprising a By “specifically binds, it is generally meant that an anti C1 domain and a C3 domain; a polypeptide chain com body binds to an epitope via its antigen binding domain, and prising a C1 domain, at least a portion of a hinge domain, 50 that the binding entails some complementarity between the and a C3 domain, or a polypeptide chain comprising a Cl antigen binding domain and the epitope. According to this domain, at least a portion of a hinge domain, a C2 domain, definition, an antibody is said to “specifically bind to an and a C3 domain. In another embodiment, a polypeptide of epitope when it binds to that epitope, via its antigen binding the invention comprises a polypeptide chain comprising a domain more readily than it would bind to a random, unre C3 domain. Further, a binding polypeptide for use in the 55 lated epitope. The term “specificity' is used herein to qualify invention may lack at least a portion of a C2 domain (e.g., all the relative affinity by which a certain antibody binds to a or part of a C2 domain). As set forth above, it will be certain epitope. For example, antibody “A” may be deemed to understood by one of ordinary skill in the art that these have a higher specificity for a given epitope than antibody domains (e.g., the heavy chain portions) may be modified “B,” or antibody “A” may be said to bind to epitope “C” with Such that they vary in amino acid sequence from the naturally 60 a higher specificity than it has for related epitope “D. occurring immunoglobulin molecule. By "preferentially binds, it is meant that the antibody In certain Sp35 antibodies, or antigen-binding fragments, specifically binds to an epitope more readily than it would variants, or derivatives thereof disclosed herein, the heavy bind to a related, similar, homologous, or analogous epitope. chain portions of one polypeptide chain of a multimer are Thus, an antibody which “preferentially binds to a given identical to those on a second polypeptide chain of the mul 65 epitope would more likely bind to that epitope than to a timer. Alternatively, heavy chain portion-containing mono related epitope, even though Such an antibody may cross mers of the invention are not identical. For example, each react with the related epitope. US 8,551,476 B2 13 14 By way of non-limiting example, an antibody may be con lent monoclonal antibody and an antigen with a highly repeat sidered to binda first epitope preferentially if it binds said first ing epitope structure, such as a polymer, would be one of high epitope with a dissociation constant (K) that is less than the avidity. antibody's K, for the second epitope. In another non-limiting Sp35 antibodies or antigen-binding fragments, variants or example, an antibody may be considered to bind a first anti derivatives thereof of the invention may also be described or gen preferentially if it binds the first epitope with an affinity specified in terms of their cross-reactivity. As used herein, the that is at least one order of magnitude less than the antibody’s term “cross-reactivity” refers to the ability of an antibody, K, for the second epitope. In another non-limiting example, specific for one antigen, to react with a second antigen; a an antibody may be considered to bind a first epitope prefer measure of relatedness between two different antigenic sub 10 stances. Thus, an antibody is cross reactive if it binds to an entially if it binds the first epitope with an affinity that is at epitope other than the one that induced its formation. The least two orders of magnitude less than the antibody's K, for cross reactive epitope generally contains many of the same the second epitope. complementary structural features as the inducing epitope, In another non-limiting example, an antibody may be con and in Some cases, may actually fit better than the original. sidered to bind a first epitope preferentially if it binds the first 15 For example, certain antibodies have some degree of cross epitope with an off rate (k(off)) that is less than the antibody's reactivity, in that they bind related, but non-identical epitopes, k(off) for the second epitope. In another non-limiting e.g., epitopes with at least 95%, at least 90%, at least 85%, at example, an antibody may be considered to bind a first least 80%, at least 75%, at least 70%, at least 65%, at least epitope preferentially if it binds the first epitope with an 60%, at least 55%, and at least 50% identity (as calculated affinity that is at least one order of magnitude less than the using methods known in the art and described herein) to a antibody's k(off) for the second epitope. In another non reference epitope. An antibody may be said to have little or no limiting example, an antibody may be considered to bind a cross-reactivity if it does not bind epitopes with less than first epitope preferentially if it binds the first epitope with an 95%, less than 90%, less than 85%, less than 80%, less than affinity that is at least two orders of magnitude less than the 75%, less than 70%, less than 65%, less than 60%, less than antibody's k(off) for the second epitope. 25 55%, and less than 50% identity (as calculated using methods An antibody or antigen-binding fragment, variant, or known in the art and described herein) to a reference epitope. derivative disclosed herein may be said to bind a target An antibody may be deemed “highly specific' for a certain polypeptide disclosed herein or a fragment or variant thereof epitope, if it does not bind any other analog, ortholog, or with an off rate (k(off)) of less than or equal to 5x10 sec', homolog of that epitope. 10 sec", 5x10 sec' or 10 sec'. More preferably, an 30 Sp35 antibodies or antigen-binding fragments, variants or antibody of the invention may be said to bind a target derivatives thereof of the invention may also be described or polypeptide disclosed herein or a fragment or variant thereof specified in terms of their binding affinity to a polypeptide of with an off rate (k(off)) less than or equal to 5x10 sec', the invention. Preferred binding affinities include those with 10 sec", 5x10 sec', or 10 sec' 5x10 sec', 10 a dissociation constant or Kd less than 5x10 M, 10M, sec, 5x107 sec' or 107 sec'. 35 5x10-3 M, 10-3M, 5x10M, 10M, 5x10-M, 10-s M, An antibody or antigen-binding fragment, variant, or 5x10M, 10M, 5x107M, 107 M., 5x10M, 10M, derivative disclosed herein may be said to bind a target 5x10M, 10M, 5x100M, 100M, 5x10M, 10''M, polypeptide disclosed herein or a fragment or variant thereof 5x10-12M, 10-12M, 5x10M, 10-13 M, 5x10M, 10 with an on rate (k(on)) of greater than or equal to 10 M' M, 5x10M, or 10.5M. sect', 5x10 M' sec, 10 M' sec' or 5x10 M' sec'. 40 Sp35 antibodies or antigen-binding fragments, variants or More preferably, an antibody of the invention may be said to derivatives thereof of the invention may be “multispecific.” bind a target polypeptide disclosed herein or a fragment or e.g., bispecific, trispecific or of greater multispecificity, variant thereof with an on rate (k(on)) greater than or equal to meaning that it recognizes and binds to two or more different 10 M' sec',5x10 M' sec 10 M' sec', or 5x10M epitopes present on one or more different antigens (e.g., pro sec' or 107M sec. 45 teins) at the same time. Thus, whether an Sp35 antibody is An antibody is said to competitively inhibit binding of a “monospecific' or “multispecific, e.g., “bispecific,” refers to reference antibody to a given epitope if it preferentially binds the number of different epitopes with which a binding to that epitope to the extent that it blocks, to some degree, polypeptide reacts. Multispecific antibodies may be specific binding of the reference antibody to the epitope. Competitive for different epitopes of a target polypeptide described herein inhibition may be determined by any method known in the art, 50 or may be specific for a target polypeptide as well as for a for example, competition ELISA assays. An antibody may be heterologous epitope, such as a heterologous polypeptide or said to competitively inhibit binding of the reference antibody Solid Support material. to a given epitope by at least 90%, at least 80%, at least 70%, As used herein the term “valency” refers to the number of at least 60%, or at least 50%. potential binding domains, e.g., antigen binding domains, As used herein, the term “affinity” refers to a measure of 55 present in an Sp35 antibody, binding polypeptide orantibody. the strength of the binding of an individual epitope with the Each binding domain specifically binds one epitope. When an CDR of an immunoglobulin molecule. See, e.g., Harlow et Sp35 antibody, binding polypeptide or antibody comprises al., Antibodies: A Laboratory Manual, (Cold Spring Harbor more than one binding domain, each binding domain may Laboratory Press, 2nd ed. 1988) at pages 27-28. As used specifically bind the same epitope, for an antibody with two herein, the term “avidity” refers to the overall stability of the 60 binding domains, termed “bivalent monospecific,” or to dif complex between a population of immunoglobulins and an ferent epitopes, for an antibody with two binding domains, antigen, that is, the functional combining strength of an termed “bivalent bispecific.” An antibody may also be bispe immunoglobulin mixture with the antigen. See, e.g., Harlow cific and bivalent for each specificity (termed “bispecific tet at pages 29-34. Avidity is related to both the affinity of indi ravalent antibodies’). In another embodiment, tetravalent vidual immunoglobulin molecules in the population with spe 65 minibodies or domain deleted antibodies can be made. cific epitopes, and also the Valencies of the immunoglobulins Bispecific bivalent antibodies, and methods of making and the antigen. For example, the interaction between a biva them, are described, for instance in U.S. Pat. Nos. 5,731, 168; US 8,551,476 B2 15 16 5,807,706; 5,821,333; and U.S. Appl. Publ. Nos. 2003/ class and preferably from an antibody from a different spe 020734 and 2002/0155537, the disclosures of all of which are cies. An engineered antibody in which one or more “donor incorporated by reference herein. Bispecific tetravalent anti CDRs from a non-human antibody of known specificity is bodies, and methods of making them are described, for grafted into a human heavy or light chain framework region is instance, in WO 02/096948 and WO 00/44788, the disclo referred to herein as a “humanized antibody.” It may not be sures of both of which are incorporated by reference herein. necessary to replace all of the CDRs with the complete CDRs See generally, PCT publications WO 93/17715; WO from the donor variable region to transfer the antigenbinding 92/08802; WO91/00360; WO92/05793: Tutt et al., J. Immu capacity of one variable domain to another. Rather, it may nol. 147:60-69 (1991): U.S. Pat. Nos. 4,474,893; 4,714,681; only be necessary to transfer those residues that are necessary 4.925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 10 to maintain the activity of the target binding site. Given the 148: 1547-1553 (1992). explanations set forth in, e.g., U.S. Pat. Nos. 5,585,089, As previously indicated, the Subunit structures and three 5,693,761, 5,693,762, and 6,180,370, it will be well within dimensional configuration of the constant regions of the vari the competence of those skilled in the art, either by carrying ous immunoglobulin classes are well known. As used herein, out routine experimentation or by trial and error testing to the term “V. domain includes the amino terminal variable 15 obtain a functional engineered or humanized antibody. domain of an immunoglobulin heavy chain and the term “C1 As used herein the term “properly folded polypeptide' domain includes the first (most amino terminal) constant includes polypeptides (e.g., Sp35 antibodies) in which all of region domain of an immunoglobulin heavy chain. The Cl the functional domains comprising the polypeptide are dis domain is adjacent to the V domain and is amino terminal to tinctly active. As used herein, the term “improperly folded the hinge region of an immunoglobulin heavy chain mol polypeptide' includes polypeptides in which at least one of ecule. the functional domains of the polypeptide is not active. In one As used herein the term “C2 domain includes the portion embodiment, a properly folded polypeptide comprises of a heavy chain molecule that extends, e.g., from about polypeptide chains linked by at least one disulfide bond and, residue 244 to residue 360 of an antibody using conventional conversely, an improperly folded polypeptide comprises numbering schemes (residues 244 to 360, Kabat numbering 25 polypeptide chains not linked by at least one disulfide bond. system; and residues 231-340, EU numbering system; see As used herein the term “engineered includes manipula Kabat EA et al. op.cit. The C2 domain is unique in that it is tion of nucleic acid or polypeptide molecules by synthetic not closely paired with another domain. Rather, two N-linked means (e.g. by recombinant techniques, in vitro peptide Syn branched carbohydrate chains are interposed between the two thesis, by enzymatic or chemical coupling of peptides or C2 domains of an intact native IgG molecule. It is also well 30 Some combination of these techniques). documented that the C3 domain extends from the C2 As used herein, the terms “linked,” “fused’ or “fusion' are domainto the C-terminal of the IgG molecule and comprises used interchangeably. These terms refer to the joining approximately 108 residues. together of two more elements or components, by whatever As used herein, the term “hinge region' includes the por means including chemical conjugation or recombinant tion of a heavy chain molecule that joins the C1 domain to 35 means. An "in-frame fusion” refers to the joining of two or the C2 domain. This hinge region comprises approximately more polynucleotide open reading frames (ORFs) to form a 25 residues and is flexible, thus allowing the two N-terminal continuous longer ORF, in a manner that maintains the cor antigen binding regions to move independently. Hinge rect translational reading frame of the original ORFs. Thus, a regions can be subdivided into three distinct domains: upper, recombinant fusion protein is a single protein containing two middle, and lower hinge domains (Roux et al., J. Immunol. 40 ore more segments that correspond to polypeptides encoded 161:4083 (1998)). by the original ORFs (which segments are not normally so As used herein the term “disulfide bond' includes the cova joined in nature.) Although the reading frame is thus made lent bond formed between two sulfur atoms. The amino acid continuous throughout the fused segments, the segments may cysteine comprises a thiol group that can form a disulfide be physically or spatially separated by, for example, in-frame bond or bridge with a second thiol group. In most naturally 45 linker sequence. For example, polynucleotides encoding the occurring IgG molecules, the C1 and C regions are linked CDRs of an immunoglobulin variable region may be fused, by a disulfide bond and the two heavy chains are linked by two in-frame, but be separated by a polynucleotide encoding at disulfide bonds at positions corresponding to 239 and 242 least one immunoglobulin framework region or additional using the Kabat numbering system (position 226 or 229, EU CDR regions, as long as the “fused CDRs are co-translated numbering system). 50 as part of a continuous polypeptide. As used herein, the term "chimeric antibody' will be held In the context of polypeptides, a "linear sequence' or a to mean any antibody wherein the immunoreactive region or "sequence' is an order of amino acids in a polypeptide in an site is obtained or derived from a first species and the constant amino to carboxyl terminal direction in which residues that region (which may be intact, partial or modified in accor neighbor each other in the sequence are contiguous in the dance with the instant invention) is obtained from a second 55 primary structure of the polypeptide. species. In preferred embodiments the target binding region The term “expression' as used herein refers to a process by or site will be from a non-human Source (e.g. mouse or pri which a gene produces a biochemical, for example, an RNA mate) and the constant region is human. or polypeptide. The process includes any manifestation of the As used herein, the term “engineered antibody' refers to an functional presence of the gene within the cell including, antibody in which the variable domain in either the heavy and 60 without limitation, gene knockdown as well as both transient light chain or both is altered by at least partial replacement of expression and stable expression. It includes without limita one or more CDRs from an antibody of known specificity and, tion transcription of the gene into messenger RNA (mRNA), if necessary, by partial framework region replacement and transfer RNA (tRNA), small hairpin RNA (shRNA), small sequence changing. Although the CDRS may be derived from interfering RNA (siRNA) or any other RNA product, and the an antibody of the same class or even Subclass as the antibody 65 translation of such mRNA into polypeptide(s). If the final from which the framework regions are derived, it is envisaged desired product is a biochemical, expression includes the that the CDRs will be derived from an antibody of different creation of that biochemical and any precursors. Expression US 8,551,476 B2 17 18 of a gene produces a “gene product.” As used herein, a gene i.e., MQVSKR (SEQID NO:3) may or may not be present at product can be either a nucleic acid, e.g., a messenger RNA the N-terminus of the Sp35 signal sequence. Table 2 lists the produced by transcription of a gene, or a polypeptide which is Sp35 domains and other regions, according to amino acid translated from a transcript. Gene products described herein residue number, based on the Sp35 amino acid sequence presented herein as SEQID NO: 2. The Sp35 polypeptide is further include nucleic acids with post transcriptional modi characterized in more detail in PCT Publication No. WO fications, e.g., polyadenylation, or polypeptides with post 2004/085648, which is incorporated herein by reference in its translational modifications, e.g., methylation, glycosylation, entirety. the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like. As used herein, the terms “treat' or “treatment” refer to 10 TABLE 2 both therapeutic treatment and prophylactic or preventative Sp35 Domains measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, Such Domain or Region Beginning Residue Ending Residue as the progression of multiple Sclerosis. Beneficial or desired Signal Sequence 1 33 or 35 clinical results include, but are not limited to, alleviation of 15 LRRNT 34 or 36 64 symptoms, diminishment of extent of disease, stabilized (i.e., LRR 66 89 LRR 90 113 not worsening) state of disease, delay or slowing of disease LRR 114 137 progression, amelioration or palliation of the disease state, LRR 138 161 and remission (whether partial or total), whether detectable or LRR 162 18S undetectable. "Treatment can also mean prolonging Survival LRR 186 209 LRR 210 233 as compared to expected Survival if not receiving treatment. LRR 234 257 Those in need of treatment include those already with the LRR 258 281 condition or disorder as well as those prone to have the LRR 282 305 condition or disorder or those in which the condition or dis LRR 306 329 25 LRR 330 353 order is to be prevented. LRRCT 363 414 or 416 By “subject” or “individual” or “animal” or “patient” or Basic 415 or 417 424 “mammal is meant any Subject, particularly a mammalian 9. 419 493 Subject, for whom diagnosis, prognosis, ortherapy is desired. Connecting sequence 494 551 Mammalian Subjects include humans, domestic animals, Transmembrane 552 576 farm animals, and Zoo, sports, or pet animals such as dogs, 30 Cytoplasmic 577 614 cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and SO. O. Tissue distribution and developmental expression of Sp35 As used herein, phrases such as “a subject that would has been studied in humans and rats. Sp35 biology has been benefit from administration of an Sp35 antibody' and “an studied in an experimental animal (rat) model. Expression of animal in need of treatment includes Subjects, such as mam 35 rat Sp35 is localized to neurons and oligodendrocytes, as malian subjects, that would benefit from administration of an determined by northern blot and immuno-histochemical Sp35 antibody used, e.g., for detection of an Sp35 polypep staining. Rat Sp35 mRNA expression level is regulated devel tide (e.g., for a diagnostic procedure) and/or from treatment, opmentally, peaking shortly after birth, i.e., ca. postnatal day i.e., palliation or prevention of a disease such as MS, with an one. In a rat spinal cord transection injury model, Sp35 is Sp35 antibody. As described in more detail herein, the Sp35 40 up-regulated at the injury site, as determined by RT-PCR. See antibody can be used in unconjugated form or can be conju Miet al. Nature Neurosci. 7:221-228 (2004). gated, e.g., to a drug, prodrug, or an isotope. In the context of the amino acids comprising the various structural and functional domains of an Sp35 polypeptide, the II. Sp35 term “about includes the particularly recited value and val 45 ues larger or smaller by several (e.g., 10,9,8,7,6, 5, 4, 3, 2, Naturally occurring human Sp35 (Sp35) is a glycosylated or 1) amino acids. Since the location of these domains as central nervous system-specific protein which is predicted to listed in Table 1 have been predicted by computer graphics, have 614 amino acids (SEQID NO: 2), including a 33 amino one of ordinary skill would appreciate that the amino acid acid signal sequence. Sp 35 is also known in the art by the residues constituting the domains may vary slightly (e.g., by names LINGO-1, LRRN6, LRRN6A, FLJ14594, LERN1, 50 about 1 to 15 residues) depending on the criteria used to MGC17422 and UNQ201. The human, full-length wild-type define the domain. Sp35 polypeptide contains an LRR domain consisting of 14 The inventors have discovered that full-length, wild-type leucine-rich repeats (including N- and C-terminal caps), an Ig Sp35 binds to NgR1. See PCT Publication No. WO 2004/ domain, a transmembrane region, and a cytoplasmic domain. 085648. The inventors have also discovered that Sp35 is The cytoplasmic domain contains a canonical tyrosine phos 55 expressed in oligodendrocytes and that the Sp35 protein is phorylation site. In addition, the naturally occurring Sp35 involved in the regulation of oligodendrocyte-mediated protein contains a signal sequence, a short basic region myelination of axons. See U.S. Patent Publication No. 2006/ between the LRRCT and Ig domain, and a transmembrane 0009388 A1, which is incorporated herein by reference in its region between the Ig domain and the cytoplasmic domain. entirety. The human Sp35 gene (SEQ ID NO:1) contains alternative 60 The nucleotide sequence for the full-length Sp35 molecule translation start codons, so that six additional amino acids, is as follows:

ATGCTGGCGGGGGGCGTGAGGAGCATGCCCAGCCCCCTCCTGGCCTGCTGGCAGCCCATCCTCCTGCTGG (SEQ ID NO: 1)

TGCTGGGCTCAGTGCTGTCAGGCTCGGCCACGGGCTGCCCGCCCCGCTGCGAGTGCTCCGCCCAGGACCG US 8,551,476 B2 19 20 - Continued CGCTGTGCTGTGCCACCGCAAGCGCTTTGTGGCAGTCCCCGAGGGCATCCCCACCGAGACGCGCCTGCTG

GACCTAGGCAAGAACCGCATCAAAACGCTCAACCAGGACGAGTTCGCCAGCTTCCCGCACCTGGAGGAGC

TGGAGCTCAACGAGAACATCGTGAGCGCCGTGGAGCCCGGCGCCTTCAACAAC CTCTTCAAC CTCCGGAC

GCTGGGTCTCCGCAGCAACCGCCTGAAGCTCATCCCGCTAGGCGTCTTCACTGGCCTCAGCAACCTGACC

AAGCTGGACATCAGCGAGAACAAGATTGTTATCCTGCTGGACTACATGTTTCAGGACCTGTACAAC CTCA

AGTCACTGGAGGTTGGCGACAATGACCTCGTCTACATCTCTCACCGCGCCTTCAGCGGCCTCAACAGCCT

GGAGCAGCTGACGCTGGAGAAATGCAACCTGACCTCCATCCCCACCGAGGCGCTGTCCCACCTGCACGGC

CTCATCGTCCTGAGGCTCCGGCACCTCAACATCAATGCCATCCGGGACTACTCCTTCAAGAGGCTCTACC

GACT CAAGGTCTTGGAGATCTCCCACTGGCCCTACTTGGACACCATGACACCCAACTGCCTCTACGGCCT

CAACCTGACGTCCCTGTCCATCACACACTGCAATCTGACCGCTGTGCCCTACCTGGCCGTCCGCCACCTA

GTCTATCTCCGCTTCCTCAAC CTCTCCTACAACCCCATCAGCACCATTGAGGGCTCCATGTTGCATGAGC

TGCTCCGGCTGCAGGAGATCCAGCTGGTGGGCGGGCAGCTGGCCGTGGTGGAGCCCTATGCCTTCCGCGG

CCTCAACTACCTGCGCGTGCTCAATGTCTCTGGCAACCAGCTGACCACACTGGAGGAATCAGTCTTCCAC

TCGGTGGGCAACCTGGAGACACTCATCCTGGACTCCAACCCGCTGGCCTGCGACTGTCGGCTCCTGTGGG

TGTTCCGGCGCCGCTGGCGGCTCAACTTCAACCGGCAGCAGCCCACGTGCGCCACGCCCGAGTTTGTCCA

GGGCAAGGAGTTCAAGGACTTCCCTGATGTGCTACTGCCCAACTACTTCACCTGCCGCCGCGCCCGCATC

CGGGACCGCAAGGCCCAGCAGGTGTTTGTGGACGAGGGCCACACGGTGCAGTTTGTGTGCCGGGCCGATG

GCGACCCGCCGCCCGCCATCCTCTGGCTCTCACCCCGAAAGCACCTGGTCTCAGCCAAGAGCAATGGGCG

GCTCACAGTCTTCCCTGATGGCACGCTGGAGGTGCGCTACGCCCAGGTACAGGACAACGGCACGTACCTG

TGCATCGCGGCCAACGCGGGCGGCAACGACTCCATGCCCGCCCACCTGCATGTGCGCAGCTACTCGCCCG

ACTGGCCCCATCAGCCCAACAAGACCTTCGCTTTCATCTCCAACCAGCCGGGCGAGGGAGAGGCCAACAG

CACCCGCGCCACTGTGCCTTTCCCCTTCGACATCAAGACCCTCATCATCGCCACCACCATGGGCTTCATC

TCTTTCCTGGGCGTCGTCCTCTTCTGCCTGGTGCTGCTGTTTCTCTGGAGCCGGGGCAAGGGCAACACAA

AGCACAACATCGAGATCGAGTATGTGCCCCGAAAGTCGGACGCAGGCATCAGCTCCGCCGACGCGCCCCG

CAAGTTCAACATGAAGATGATATGA.

The polypeptide sequence for the full-length Sp35 monoclonal antibodies, and fragments, variants, and deriva polypeptide is as follows: tives thereof shown in Tables 3A and 3B.

MLAGGVRSMPSPLLACWOPILLLVLGSVLSGSATGCPPRCECSAODRAVLCHRKRFVAVPEGIPTETRL (SEQ ID NO: 2) LDLGKNRIKTLNODEFASFPHLEELELNENIVSAVEPGAFNNLFNLRTLGLRSNRLKLIPLGVFTGLSN

LTKLDISENKIVILLDYMFODLYNLKSLEVGDNDLVYISHAAFSGLNSLEOLTLEKCNLTSIPTEALSH

LHGLIWLRLRHLNINAIRDYSFKRLYRLKWLEISHWPYLDTMTPNCLYGLNLTSLSITHCNLTAWPYLA

WRHLVYLRFLNLSYNPISTIEGSMLHELLRLOEIOLVGGOLAVWEPYAFRGLNYLRVLNVSGNOLTTLE

ESVFHSVGNLETLILDSNPLACDCRLLWWFRRRWRLNFNROOPTCATPEFVOGKEFAAFPDVLLPNYFT

CRRARIRDRKAOOVFWDEGHTWOFVCRADGDPPPAILWLSPRKHLVSAKSNGRLTVFPDGTLEVRYAOV

ODNGTYLCIAANAGGNDSMPAHLHVRSYSPDWPHOPNKTFAFISNOPGEGEANSTRATVPFPFDIKTLI

IATTMGFISFLGWWLFCLWLLFLWSRGKGNTKHIEIEYWPRKSDAGISSADAPRKNMKMI.H 60 III. Sp35 Antibodies Table 3A describes the regions of the Sp35 polypeptide that are bound by certain full-length phage library derived anti In one embodiment, the present invention is directed to bodies. These antibodies have the same variable regions as the Sp35 antibodies, or antigen-binding fragments, variants, or 65 Fab fragments derived from Phage Display Library-1, as indi derivatives thereof. For example, the present invention cated in Table 3B (e.g. D05 in Table 3A has the same variable includes at least the antigen-binding domains of certain region as Li05 in Table 3B, D06 in Table 3A has the same US 8,551,476 B2 21 22 variable region as Li06 in Table 3B, etc.). The antibodies were assays are described herein or are well known and understood tested for binding Sp35 fragments as defined in Table 3A, by those of ordinary skill in the art. Hybridoma-derived using methods well known in the art. monoclonal antibodies listed in Table 3B were produced by Table 3B describes the ability of the named monoclonal antibodies or Fab fragments to detect Sp35 in various assays injection of soluble Sp35 into mice and then isolated using such as: Fluorescent Activated Cell Sorting (FACS), Immu hybridoma technology which is well known in the art and noprecipitation (IP), Western blot analysis, Immunohis described herein. Monoclonal antibodies and antibody Fab tochemistry (IHC) and Enzyme Linked Immunosorbent fragments listed in Table 3B were isolated from two different Assay (ELISA). Detailed protocols for performing these phage display libraries using techniques known in the art. TABLE 3A

DO3 D05 DO6 D08 D11 D13 D33 (LiO3 (LiO5 (LiO6 (LiO8 (LiO3 (Li13 (Li33 Sp35 Variable Variable Variable Variable Variable Variable Variable Fragment Region) Region) Region) Region) Region) Region) Region)

1-432 rat ------FC 417-493 +f- +f- rat Fo AP-Sp35 ND ------ND ND ND (1-419) AP-Sp35 ND ND ND NID (418-498) 417-498 human Fc 417-SO3 human Fc 363-498 human Fc 244-498 human Fc

US 8,551,476 B2 29 30 As used herein, the term “antigen binding domain' region); 417 to 493: 417 to 532: 419 to 493 (the Sp351 g includes a site that specifically binds an epitope on an antigen region); or 425 to 532 of SEQID NO:2. (e.g., an epitope of Sp35). The antigen binding domain of an Additional Sp35 peptide fragments to which certain anti antibody typically includes at least a portion of an immuno bodies, or antigen-binding fragments, variants, or derivatives globulin heavy chain variable region and at least a portion of 5 thereof of the present invention bind include, but are not an immunoglobulin light chain variable region. The binding limited to those fragments comprising, consisting essentially site formed by these variable regions determines the specific of or consisting of one or more leucine-rich-repeats (LRR) of ity of the antibody. Sp35. Such fragments, include, for example, fragments com The present invention is more specifically directed to an prising, consisting essentially of, or consisting of amino acids Sp35 antibody, or antigen-binding fragment, variant or 10 derivatives thereof, where the Sp35 antibody binds to the 66 to 89: 66 to 113: 66 to 137; 90 to 113; 114 to 137: 138 to same epitope as a monoclonal antibody selected from the 161: 162 to 185; 186 to 209; 210 to 233: 234 to 257; 258 to group consisting of 201'. 3A3, 3A6, 1A7, 1G7. 2B10, 2C11. 281; 282 to 305:306 to 329; or 330 to 353 of SEQID NO:2. 2F3, 3P1D10.2C3, 3P1E1 1.3B7, 3P2C6.3G10.2H7, Corresponding fragments of a variant Sp35 polypeptide at 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 15 least 70%, 75%, 80%, 85%, 90%, or 95% identical to amino 3P4C5.1 D8, 3P4C8.2G9, 30-C12 (LiO1), 38-D01 (LiO2), acids 66 to 89: 66 to 113: 90 to 113; 114 to 137: 138 to 161; 35-E04 (LiO3), 36-C09 (LiO4), 30-A11 (LiO5), 34-F02 162 to 185; 186 to 209; 210 to 233: 234 to 257; 258 to 281; (LiO6), 29-E07 (LiO7), 34-G04 (LiO8), 36-A 12 (LiO9), 282 to 305:306 to 329; or 330 to 353 of SEQID NO:2 are also 28-D02 (Li10), 30-B01 (Li11), 34-B03 (Li12), Li13, Li32, contemplated. Li33, Li34, 3383 (L1a.1), 3495 (L1 a.2), 3563 (L1 a.3), 3564 Additional Sp35 peptide fragments to which certain anti (L1 a.4), 3565 (L1 a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 bodies, or antigen-binding fragments, variants, or derivatives (L1a.8), 3569 (L1a.9), 3570 (L1a. 10), 3571 (L1a. 11), 3582 thereof of the present invention bind include, but are not (L1a. 12), and 1968 (L1a.13). limited to those fragments comprising, consisting essentially The invention is further drawn to an Sp35 antibody, or of or consisting of one or more cysteine rich regions flanking antigen-binding fragment, variant or derivatives thereof, 25 the LRR of Sp35. Such fragments, include, for example, a where the Sp35 antibody competitively inhibits a monoclonal fragment comprising, consisting essentially of or consisting antibody selected from the group consisting of 201'. 3A3, of amino acids 34 to 64 of SEQID NO:2 (the N-terminal LRR 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3, flanking region (LRRNT)), or a fragment comprising, con 3P1E1 1.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, sisting essentially of or consisting of amino acids 363 to 416 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1 D8, 3P4C8.2G9, 30-C12 30 of SEQID NO:2 (the C-terminal LRR flanking region (LR (L100), 38-D01 (LiO2), 35-E04 (LiO3), 36-C09 (LiO4), RCT)), amino acids Corresponding fragments of a variant 30-A11 (Li05), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 Sp35 polypeptide at least 70%, 75%, 80%, 85%, 90%, or 95% (LiO8), 36-A12 (LiO9), 28-D02 (Li10), 30-B01 (Li11), identical to amino acids 34 to 64 and 363 to 416 of SEQID 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495 NO:2 are also contemplated. (L1 a.2), 3563 (L1 a.3), 3564 (L1 a.4), 3565 (L1a.5), 3566 35 As known in the art, “sequence identity” between two (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 polypeptides is determined by comparing the amino acid (L1a. 10), 3571 (L1a. 11), 3582 (L1a. 12), and 1968 (L1a.13) sequence of one polypeptide to the sequence of a second from binding to Sp35. polypeptide. When discussed herein, whether any particular The invention is also drawn to an Sp35 antibody, or anti polypeptide is at least about 70%, 75%, 80%, 85%, 90% or gen-binding fragment, variant or derivatives thereof, where 40 95% identical to another polypeptide can be determined using the Sp35 antibody comprises at least the antigen binding methods and computer programs/software known in the art region of a monoclonal antibody selected from the group such as, but not limited to, the BESTFIT program (Wisconsin consisting of 201', 3A3, 3A6, 1A7, 1G7. 2B10, 2011, 2F3, Sequence Analysis Package, Version 8 for Unix, Genetics 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, Computer Group, University Research Park, 575 Science 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1 D8, 45 Drive, Madison, Wis. 53711). BESTFIT uses the local 3P4C8.2G9, 30-C12 (L100), 38-D01 (LiO2), 35-E04 (LiO3), homology algorithm of Smith and Waterman, Advances in 36-C09 (LiO4), 30-A11 (LiO5), 34-F02 (LiO6), 29-E07 Applied Mathematics 2:482-489 (1981), to find the best seg (LiO7), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), ment of homology between two sequences. When using 30-B01 (Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34,3383 BESTFIT or any other sequence alignment program to deter (L1a.1), 3495 (L1 a.2), 3563 (L1 a.3), 3564 (Lla.4), 3565 50 mine whether a particular sequence is, for example, 95% (L1 a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569 identical to a reference sequence according to the present (L1a.9), 3570 (L1a. 10), 3571 (L1a. 11), 3582 (L1a.12), and invention, the parameters are set, of course, Such that the 1968 (L1a.13). percentage of identity is calculated over the full length of the In certain embodiments, the present invention is directed to reference polypeptide sequence and that gaps in homology of an antibody, or antigen-binding fragment, variant, or deriva 55 up to 5% of the total number of amino acids in the reference tive thereof which specifically or preferentially binds to a sequence are allowed. particular Sp35 polypeptide fragment or domain. Such Sp35 Additional Sp35 peptide fragments to which certain anti polypeptide fragments include, but are not limited to, an Sp35 bodies, or antigen-binding fragments, variants, or derivatives polypeptide comprising, consisting essentially of, or consist thereof of the present invention bind include, but are not ing of amino acids 34 to 532: 34 to 417:34 to 425; 34 to 493; 60 limited to those fragments comprising, consisting essentially 66 to 532: 66 to 417; 66 to 426; 66 to 493: 66 to 532; 417 to of, or consisting of amino acids 41 to 525 of SEQID NO:2:40 532: 417 to 425 (the Sp35 basic region); 417 to 493: 417 to to 526 of SEQIDNO:2:39 to 527 of SEQIDNO:2:38 to 528 532:419 to 493 (the Sp351 g region); or 425 to 532 of SEQID of SEQ ID NO:2; 37 to 529 of SEQID NO:2: 36 to 530 of NO:2; or an Sp35 variant polypeptide at least 70%, 75%, SEQID NO:2:35 to 531 of SEQID NO:2:34 to 531 of SEQ 80%, 85%, 90%, or 95% identical to amino acids 34 to 532: 65 ID NO:2; 46 to 520 of SEQID NO:2: 45 to 521 of SEQ ID 34 to 417:34 to 425:34 to 493; 66 to 532: 66 to 417; 66 to 426; NO:2:44 to 522 of SEQID NO:2:43 to 523 of SEQID NO:2: 66 to 493; 66 to 532: 417 to 532: 417 to 425 (the Sp35 basic and 42 to 524 of SEQID NO:2. US 8,551,476 B2 31 32 Still additional Sp35 peptide fragments to which certain NO:2:36 to 305 of SEQID NO:2:36 to 329 of SEQID NO:2: antibodies, orantigen-binding fragments, variants, or deriva 36 to 353 of SEQID NO:2:36 to 416 of SEQID NO:2:36 to tives thereof of the present invention bind include, but are not 424 of SEQID NO:2: 36 to 493 of SEQID NO:2; and 36 to limited to those fragments comprising, consisting essentially 551 of SEQID NO:2. of, or consisting of amino acids 1 to 33 of SEQID NO:2; 1 to 5 Additional Sp35 peptide fragments to which certain anti 35 of SEQ ID NO:2: 34 to 64 of SEQID NO:2: 36 to 64 of bodies, or antigen-binding fragments, variants, or derivatives SEQID NO:2: 66 to 89 of SEQID NO:2:90 to 113 of SEQID thereof of the present invention bind include, but are not NO:2; 114 to 137 of SEQID NO:2: 138 to 161 of SEQ ID limited to those fragments comprising, consisting essentially NO:2; 162 to 185 of SEQID NO:2: 186 to 209 of SEQ ID of, or consisting of amino acids 36 to 530 of SEQID NO:2:36 NO:2: 210 to 233 of SEQID NO:2: 234 to 257 of SEQ ID 10 to 531 of SEQID NO:2:36 to 532 of SEQID NO:2:36 to 533 NO:2: 258 to 281 of SEQID NO:2: 282 to 305 of SEQ ID of SEQID NO:2: 36 to 534 of SEQ ID NO:2: 36 to 535 of NO:2: 306 to 329 of SEQ ID NO:2: 330 to 353 of SEQ ID SEQID NO:2:36 to 536 of SEQID NO:2:36 to 537 of SEQ NO:2: 363 to 416 of SEQID NO:2: 417 to 424 of SEQ ID ID NO:2:36 to 538 of SEQID NO:2; and 36 to 539 of SEQ NO:2:419 to 493 of SEQID NO:2; and 494 to 551 of SEQID ID NO:2. NO:2. 15 More Sp35 peptide fragments to which certain antibodies, Further still, Sp35 peptide fragments to which certain anti or antigen-binding fragments, variants, or derivatives thereof bodies, or antigen-binding fragments, variants, or derivatives of the present invention bind include, but are not limited to thereof of the present invention bind include, but are not those fragments comprising, consisting essentially of, or con limited to those fragments comprising, consisting essentially sisting of amino acids 417 to 493 of SEQID NO:2:417 to 494 of, or consisting of amino acids 1 to 33 of SEQID NO:2; 1 to of SEQID NO:2:417 to 495 of SEQID NO:2:417 to 496 of 35 of SEQID NO:2:1 to 64 of SEQID NO:2:1 to 89 of SEQ SEQID NO:2:417 to 497 of SEQ ID NO:2: 417 to 498 of IDNO:2:1 to 113 of SEQID NO:2:1 to 137 of SEQID NO:2: SEQID NO:2:417 to 499 of SEQ ID NO:2: 417 to 500 of 1 to 161 of SEQID NO:2; 1 to 185 of SEQID NO:2:1 to 209 SEQID NO:2:417 to 492 of SEQ ID NO:2: 417 to 491 of of SEQID NO:2; 1 to 233 of SEQID NO:2:1 to 257 of SEQ SEQID NO:2:412 to 493 of SEQ ID NO:2: 413 to 493 of IDNO:2:1 to 281 of SEQID NO:2:1 to 305 of SEQID NO:2: 25 SEQID NO:2:414 to 493 of SEQ ID NO:2: 415 to 493 of 1 to 329 of SEQID NO:2; 1 to 353 of SEQID NO:2:1 to 416 SEQID NO:2: 416 to 493 of SEQ ID NO:2; 411 to 493 of of SEQID NO:2; 1 to 424 of SEQID NO:2:1 to 493 of SEQ SEQID NO:2:410 to 493 of SEQ ID NO:2: 410 to 494 of IDNO:2:1 to 551 of SEQID NO:2:1 to 531 of SEQID NO:2 SEQID NO:2: 411 to 494 of SEQ ID NO:2: 412 to 494 of and 1 to 532 of SEQID NO:2. SEQID NO:2:413 to 494 of SEQ ID NO:2: 414 to 494 of Additional Sp35 peptide fragments to which certain anti 30 SEQID NO:2:415 to 494 of SEQ ID NO:2: 416 to 494 of bodies, or antigen-binding fragments, variants, or derivatives SEQID NO:2:417 to 494 of SEQID NO:2; and 418 to 494 of thereof of the present invention bind include, but are not SEQID NO:2. limited to those fragments comprising, consisting essentially In an additional embodiment Sp35 peptide fragments to of, or consisting of amino acids 34 to 64 of SEQID NO:2: 34 which certain antibodies, or antigen-binding fragments, Vari to 89 of SEQID NO:2: 34 to 113 of SEQID NO:2: 34 to 137 35 ants, or derivatives thereof of the present invention bind of SEQID NO:2: 34 to 161 of SEQ ID NO:2: 34 to 185 of include, an Sp35 polypeptide comprising, consisting essen SEQID NO:2: 34 to 209 of SEQID NO:2: 34 to 233 of SEQ tially of, or consisting of peptides of the Ig domain of Sp35 or ID NO:2: 34 to 257 of SEQID NO:2: 34 to 281 of SEQ ID fragments, variants, or derivatives of Such polypeptides. Spe NO:2:34 to 305 of SEQID NO:2:34 to 329 of SEQID NO:2: cifically, polypeptides comprising, consisting essentially of 34 to 353 of SEQID NO:2: 34 to 416 of SEQID NO:2: 34 to 40 or consisting of the following polypeptide sequences: 424 of SEQID NO:2: 34 to 493 of SEQID NO:2; and 34 to ITXXX (SEQ ID NO:287), ACXXX (SEQ ID 551 of SEQID NO:2. NO:288), VCXXX (SEQ ID NO:289) and SPXXX, More additional Sp35 peptide fragments to which certain (SEQ ID NO:290) where X is lysine, arginine, histidine, antibodies, orantigen-binding fragments, variants, or deriva glutamine, or asparagine, X2 is lysine, arginine, histidine, tives thereof of the present invention bind include, but are not 45 glutamine, or asparagine and X is lysine, arginine, histidine, limited to those fragments comprising, consisting essentially glutamine, or asparagine. For example, Sp35 peptide frag of, or consisting of amino acids 34 to 530 of SEQID NO:2: 34 ments to which certain antibodies, or antigen-binding frag to 531 of SEQID NO:2:34 to 532 of SEQID NO:2:34 to 533 ments, variants, or derivatives thereof of the present invention of SEQID NO:2: 34 to 534 of SEQ ID NO:2: 34 to 535 of bind include, those fragments comprising, consisting essen SEQID NO:2: 34 to 536 of SEQID NO:2: 34 to 537 of SEQ 50 tially of, or consisting of the following polypeptide ID NO:2: 34 to 538 of SEQID NO:2: 34 to 539 of SEQ ID sequences: SPRKH (SEQ ID NO:291), SPRKK (SEQ ID NO:2:30 to 532 of SEQIDNO:2:31 to 532 of SEQID NO:2; NO:292), SPRKR (SEQ ID NO:293), SPKKH (SEQ ID 32 to 532 of SEQID NO:2:33 to 532 of SEQID NO:2: 34 to NO:294), SPHKH (SEQ ID NO:295), SPRRH (SEQ ID 532 of SEQID NO:2:35 to 532 of SEQID NO:2:36 to 532 NO:296), SPRHH (SEQ ID NO:297), SPRRR (SEQ ID of SEQID NO:2:30 to 531 of SEQ ID NO:2; 31 to 531 of 55 NO:298), SPHHH (SEQ ID NO:299) SPKKK (SEQ ID SEQID NO:2:32 to 531 of SEQID NO:2:33 to 531 of SEQ NO:300), LSPRKH (SEQ ID NO:301), LSPRKK (SEQ ID ID NO:2: 34 to 531 of SEQID NO:2: 35 to 531 of SEQ ID NO:302), LSPRKR (SEQ ID NO:303), LSPKKH (SEQ ID NO:2; and 36 to 531 of SEQID NO:2. NO:304), LSPHKH (SEQ ID NO:305), LSPRRH (SEQ ID Further still, Sp35 peptide fragments to which certain anti NO:306), LSPRHH (SEQ ID NO:307), LSPRRR (SEQ ID bodies, or antigen-binding fragments, variants, or derivatives 60 NO:308), LSPHHH (SEQ ID NO:309) LSPKKK (SEQ ID thereof of the present invention bind include, but are not NO:310), WLSPRKH (SEQID NO:311), WLSPRKK (SEQ limited to those fragments comprising, consisting essentially ID NO:312), WLSPRKR (SEQ ID NO:313), WLSPKKH of, or consisting of amino acids 36 to 64 of SEQID NO:2: 36 (SEQ ID NO:314), WLSPHKH (SEQ ID NO:315), to 89 of SEQID NO:2:36 to 113 of SEQID NO:2:36 to 137 WLSPRRH (SEQ ID NO:316), WLSPRHH (SEQ ID of SEQID NO:2: 36 to 161 of SEQ ID NO:2: 36 to 185 of 65 NO:317), WLSPRRR (SEQID NO:318), WLSPHHH (SEQ SEQID NO:2:36 to 209 of SEQID NO:2:36 to 233 of SEQ ID NO:319). WLSPKKK (SEQ ID NO:320). These Sp35 ID NO:2: 36 to 257 of SEQID NO:2: 36 to 281 of SEQ ID polypeptides include the basic “RKH loop' (Arginine US 8,551,476 B2 33 34 Lysine-Histidine amino acids 456-458) in the Ig domain of in Morris, G. Epitope Mapping Protocols, New Jersey: Sp35. Additional Sp35 peptides which include a basic trip Humana Press (1996), which are both incorporated herein by eptide are ITPKRR (SEQ ID NO:321), ACHHK (SEQ ID reference in their entireties. Epitope mapping can also be NO:322) and VCHHK (SEQID NO:323). performed by commercially available means (i.e. Proto Additional Sp35 peptide fragments to which certain anti PROBE, Inc. (Milwaukee, Wis.)). bodies, or antigen-binding fragments, variants, or derivatives Additionally, antibodies produced which bind to any por thereof of the present invention bind include, an Sp35 tion of Sp35 can then be screened for their ability to act as an polypeptide comprising, consisting essentially of, or consist antagonist of Sp35 and thus promote neurite outgrowth, neu ing of peptides of the Ig domain of Sp35 or fragments, vari ronal and oligodendrocyte survival, proliferation and differ ants, or derivatives of Such polypeptides. Specifically, pep 10 entiation as well as promote myelination. Antibodies can be tides comprising, consisting essentially of, or consisting of screened for oligodendrocyte/neuronal Survival by using the the following polypeptide sequences: XXRKH (SEQ ID method as described in Examples 10 and 11. Additionally, NO:324), XXRRR (SEQID NO:325), XXKKK (SEQID antibodies can be screened for their ability to promote myeli NO:326), XXHHH(SEQIDNO:327), XXRKK (SEQID nation by using the method of Example 9. Finally, antibodies NO:328), XXRKR (SEQID NO:329), XXKKH (SEQID 15 can be screened for their ability to promote oligodendrocyte NO:330), XXHKH (SEQIDNO:331), XXRRH(SEQID proliferation and differentiation, as well as neurite outgrowth NO:332) and XXRHH (SEQID NO:333) where X is any by using the method as described in Example 7. Otherantago amino acid and Xs is any amino acid. nist functions of antibodies of the present invention can be In other embodiments Sp35 peptide fragments to which tested using other assays as described in the Examples herein. certain antibodies, or antigen-binding fragments, variants, or In other embodiments, the present invention includes an derivatives thereof of the present invention bind include, an antibody, or antigen-binding fragment, variant, or derivative Sp35 polypeptide comprising, consisting essentially of, or thereof which specifically or preferentially binds to at least consisting of peptides of the Ig domain of Sp35 or fragments, one epitope of Sp35, where the epitope comprises, consists variants, or derivatives of Such polypeptides. Specifically, essentially of, or consists of at least about four to five amino polypeptides comprising, consisting essentially of, or con 25 acids of SEQID NO:2, at least seven, at least nine, or between sisting of the following polypeptide sequences: ITXXXs at least about 15 to about 30 amino acids of SEQ ID NO:2. (SEQ ID NO:334), ACXXXs (SEQ ID NO:335), The amino acids of a given epitope of SEQ ID NO:2 as VCXXX (SEQ ID NO:336) and SPXXX (SEQ ID described may be, but need not be contiguous or linear. In NO:337) where X is lysine, arginine, histidine, glutamine, or certain embodiments, the at least one epitope of Sp35 com asparagine, X-7 is any amino acid and Xs is lysine, arginine, 30 prises, consists essentially of, or consists of a non-linear histidine, glutamine, or asparagine. For example, a polypep epitope formed by the extracellular domain of Sp35 as tide comprising, consisting essentially of, or consisting of the expressed on the surface of a cell or as a soluble fragment, following polypeptide sequence: SPRLH (SEQID NO:338). e.g., fused to an IgGFc region. Thus, in certain embodiments Sp35 peptide fragments to which certain antibodies, or the at least one epitope of Sp35 comprises, consists essen antigen-binding fragments, variants, or derivatives thereof of 35 tially of, or consists of at least 4, at least 5, at least 6, at least the present invention bind include, an Sp35 polypeptide com 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least prising, consisting essentially of, or consisting of peptides 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, which contain amino acids 452-458 in the Ig domain of Sp35, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 orderivatives thereof, whereinamino acid 452 is a tryptophan contiguous or non-contiguous amino acids of SEQID NO:2. or phenylalanine residue. 40 where the non-contiguous amino acids form an epitope Additional Sp35 peptide fragments to which certain anti through protein folding. bodies, or antigen-binding fragments, variants, or derivatives In other embodiments, the present invention includes an thereof of the present invention bind include, an Sp35 antibody, or antigen-binding fragment, variant, or derivative polypeptide comprising, consisting essentially of, or consist thereof which specifically or preferentially binds to at least ing of peptides of the basic domain of Sp35. Specifically, 45 one epitope of Sp35, where the epitope comprises, consists peptides comprising, consisting essentially of, or consisting essentially of, or consists of, in addition to one, two, three, of the following polypeptide sequences: RRARIRDRK (SEQ four, five, six or more contiguous or non-contiguous amino ID NO:339), KKVKVKEKR (SEQ ID NO:340), RRLRL acids of SEQID NO:2 as described above, and an additional RDRK (SEQID NO:341), RRGRGRDRK (SEQID NO:342) moiety which modifies the protein, e.g., a carbohydrate moi and RRIRARDRK (SEQ ID NO:343). 50 ety may be included such that the Sp35 antibody binds with Additional exemplary soluble Sp35 polypeptides and higher affinity to modified target protein than it does to an methods and materials for obtaining these molecules for pro unmodified version of the protein. Alternatively, the Sp35 ducing antibodies or antibody fragments of the present inven antibody does not bind the unmodified version of the target tion may be found, e.g., in International Patent Application protein at all. No. PCT/US2004/008323, incorporated herein by reference 55 In certain aspects, the present invention is directed to an in its entirety. antibody, or antigen-binding fragment, variant, or derivative Methods of making antibodies are well known in the art thereof which specifically binds to a Sp35 polypeptide or and described herein. Once antibodies to various fragments fragment thereof, or an Sp35 variant polypeptide, with an of, or to the full-length Sp35 without the signal sequence, affinity characterized by a dissociation constant(K) which is have been produced, determining which amino acids, or 60 less than the K for said reference monoclonal antibody. epitope, of Sp35 to which the antibody or antigen binding In certain embodiments, an antibody, or antigen-binding fragment binds can be determined by eptiope mapping pro fragment, variant, or derivative thereof of the invention binds tocols as described herein as well as methods known in the art specifically to at least one epitope of Sp35 or fragment or (e.g. double antibody-sandwich ELISA as described in variant described above, i.e., binds to Such an epitope more “Chapter 11—Immunology. Current Protocols in Molecular 65 readily than it would bind to an unrelated, or random epitope; Biology, Ed. Ausubel et al., v.2, John Wiley & Sons, Inc. binds preferentially to at least one epitope of Sp35 or frag (1996)). Additional epitope mapping protocols may be found ment or variant described above, i.e., binds to such an epitope US 8,551,476 B2 35 36 more readily than it would bind to a related, similar, homolo polypeptide disclosed herein, e.g., Sp35. In another embodi gous, or analogous epitope; competitively inhibits binding of ment, a bispecific Sp35 antibody, binding polypeptide, or a reference antibody which itself binds specifically or prefer antibody has at least one binding domain specific for an entially to a certain epitope of Sp35 or fragment or variant epitope on a target polypeptide and at least one target binding described above; or binds to at least one epitope of Sp35 or domain specific for a drug or toxin. In yet another embodi fragment or variant described above with an affinity charac ment, a bispecific Sp35 antibody, binding polypeptide, or terized by a dissociation constant K, of less than about antibody has at least one binding domain specific for an 5x10 M, about 10 M, about 5x10 M, about 10 M, epitope on a target polypeptide disclosed herein, and at least about 5x10M, about 10 M, about 5x10 M, about 10 one binding domain specific for a prodrug. A bispecific Sp35 M, about 5x10M, about 10 M, about 5x107M, about 10 antibody, binding polypeptide, or antibody may be a tetrava 107 M, about 5x10 M, about 10 M, about 5x10 M, lent antibody that has two target binding domains specific for about 10 M, about 5x109 M, about 109 M, about 5x an epitope of a target polypeptide disclosed herein and two 10' M, about 10' M, about 5x10' M, about 10' M, target binding domains specific for a second target. Thus, a about 5x10' M, about 10 M, about 5x10' M, about tetravalent bispecific Sp35 antibody, binding polypeptide, or 10' M, about 5x10' M, or about 10 M. In a particular 15 antibody may be bivalent for each specificity. aspect, the antibody or fragment thereof preferentially binds Sp35 antibodies, orantigen-binding fragments, variants, or to a human Sp35 polypeptide or fragment thereof, relative to derivatives thereof of the invention, as known by those of a murine Sp35 polypeptide or fragment thereof. ordinary skill in the art, can comprise a constant region which As used in the context of antibody binding dissociation mediates one or more effector functions. For example, bind constants, the term “about allows for the degree of variation ing of the C1 component of complement to an antibody con inherent in the methods utilized for measuring antibody affin stant region may activate the complement system. Activation ity. For example, depending on the level of precision of the of complement is important in the opsonisation and lysis of instrumentation used, standard error based on the number of cell pathogens. The activation of complement also stimulates samples measured, and rounding error, the term “about 10 the inflammatory response and may also be involved in M” might include, for example, from 0.05 M to 0.005 M. 25 autoimmune hypersensitivity. Further, antibodies bind to In specific embodiments, an antibody, or antigen-binding receptors on various cells via the Fc region, with a Fc receptor fragment, variant, or derivative thereof of the invention binds binding site on the antibody Fc region binding to a Fc receptor Sp35 polypeptides or fragments or variants thereof with an (FcR) on a cell. There are a number of Fc receptors which are off rate (k(off)) of less than or equal to 5x10° sec', 10° specific for different classes of antibody, including IgG sec", 5x10 sec' or 10 sec'. Alternatively, an antibody, 30 (gamma receptors), IgE (epsilon receptors), IgA (alpha or antigen-binding fragment, variant, or derivative thereof of receptors) and IgM (mu receptors). Binding of antibody to Fc the invention binds Sp35 polypeptides or fragments or vari receptors on cell surfaces triggers a number of important and ants thereof with an off rate (k(off)) of less than or equal to diverse biological responses including engulfment and 5x10 sec', 10 sec, 5x10 sec', or 10 sec' 5x10 destruction of antibody-coated particles, clearance of sec', 10 sec, 5x107 sec' or 107 sec'. 35 immune complexes, lysis of antibody-coated target cells by In other embodiments, an antibody, or antigen-binding killer cells (called antibody-dependent cell-mediated cyto fragment, variant, or derivative thereof of the invention binds toxicity, or ADCC), release of inflammatory mediators, pla Sp35 polypeptides or fragments or variants thereof with an on cental transfer and control of immunoglobulin production. rate (k(on)) of greater than or equal to 10 M' sec", 5x10 Accordingly, certain embodiments of the invention include M' sec', 10 M' sec' or 5x10 M' sec'. Alternatively, 40 an Sp35 antibody, or antigen-binding fragment, variant, or an antibody, or antigen-binding fragment, variant, or deriva derivative thereof, in which at least a fraction of one or more tive thereof of the invention binds Sp35 polypeptides or frag of the constant region domains has been deleted or otherwise ments or variants thereof with an on rate (k(on)) greater than altered so as to provide desired biochemical characteristics or equal to 10 M' sec',5x10M sec', 10 M' sec', or Such as reduced effector functions, the ability to non-co 5X106 M sec' or 107M sec. 45 valently dimerize, increased ability to localize at the site of a In various embodiments, an Sp35 antibody, or antigen tumor, reduced serum half-life, or increased serum half-life binding fragment, variant, or derivative thereofas described when compared with a whole, unaltered antibody of approxi herein is an antagonist of Sp35 activity. In certain embodi mately the same immunogenicity. For example, certain anti ments, for example, binding of an antagonist Sp35 antibody bodies for use in the diagnostic and treatment methods to Sp35, as expressed on neurons, blocks myelin-associated 50 described herein are domain deleted antibodies which com neurite outgrowth inhibition or neuronal cell death. In other prise a polypeptide chain similar to an immunoglobulin heavy embodiments, binding of the Sp35 antibody to Sp35, as chain, but which lack at least a portion of one or more heavy expressed on oligodendrocytes, blocks inhibition of oligo chain domains. For instance, in certain antibodies, one entire dendrocyte growth or differentiation, or blocks demyelina domain of the constant region of the modified antibody will tion or dysmyelination of CNS neurons. 55 be deleted, for example, all or part of the C2 domain will be Unless it is specifically noted, as used herein a "fragment deleted. thereof in reference to an antibody refers to an antigen In certain Sp35 antibodies, or antigen-binding fragments, binding fragment, i.e., a portion of the antibody which spe variants, or derivatives thereof described herein, the Fc por cifically binds to the antigen. In one embodiment, an Sp35 tion may be mutated to decrease effector function using tech antibody, e.g., an antibody of the invention is a bispecific 60 niques known in the art. For example, the deletion or inacti Sp35 antibody, binding polypeptide, or antibody, e.g., a Vation (through point mutations or other means) of a constant bispecific antibody, minibody, domain deleted antibody, or region domain may reduce Fc receptor binding of the circu fusion protein having binding specificity for more than one lating modified antibody thereby increasing tumor localiza epitope, e.g., more than one antigen or more than one epitope tion. In other cases it may be that constant region modifica on the same antigen. In one embodiment, a bispecific Sp35 65 tions consistent with the instant invention moderate antibody, binding polypeptide, or antibody has at least one complement binding and thus reduce the serum half life and binding domain specific for at least one epitope on a target nonspecific association of a conjugated cytotoxin. Yet other US 8,551,476 B2 37 38 modifications of the constant region may be used to modify ous methods, including (a) grafting the entire non-human disulfide linkages or oligosaccharide moieties that allow for variable domains onto human constant regions to generate enhanced localization due to increased antigen specificity or chimeric antibodies; (b) grafting at least a part of one or more antibody flexibility. The resulting physiological profile, bio of the non-human complementarity determining regions availability and other biochemical effects of the modifica (CDRS) into a human framework and constant regions with or tions, such as tumor localization, biodistribution and serum without retention of critical framework residues; or (c) trans half-life, may easily be measured and quantified using well planting the entire non-human variable domains, but "cloak know immunological techniques without undue experimen ing them with a human-like section by replacement of Sur tation. face residues. Such methods are disclosed in Morrison et al., Modified forms of Sp35 antibodies, or antigen-binding 10 Proc. Natl. Acad. Sci. 81:6851-6855 (1984); Morrison et al., fragments, variants, orderivatives thereof of the invention can Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science be made from whole precursor or parent antibodies using 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 techniques known in the art. Exemplary techniques are dis (1991); Padlan, Molec. Immun. 31.169-217 (1994), and U.S. cussed in more detail herein. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all In certain embodiments both the variable and constant 15 of which are hereby incorporated by reference in their regions of Sp35 antibodies, or antigen-binding fragments, entirety. variants, or derivatives thereofare fully human. Fully human De-immunization can also be used to decrease the immu antibodies can be made using techniques that are known in the nogenicity of an antibody. As used herein, the term “de art and as described herein. For example, fully human anti immunization includes alteration of an antibody to modifyT bodies against a specific antigen can be prepared by admin cell epitopes (see, e.g., WO9852976A1, WO0034317A2). istering the antigen to a transgenic animal which has been For example, V, and V, sequences from the starting antibody modified to produce Such antibodies in response to antigenic are analyzed and a human T cell epitope 'map' from each V challenge, but whose endogenous loci have been disabled. region showing the location of epitopes in relation to comple Exemplary techniques that can be used to make such antibod mentarity-determining regions (CDRS) and other key resi ies are described in U.S. Pat. Nos. 6,150,584; 6,458,592: 25 dues within the sequence. Individual T cell epitopes from the 6.420,140 which are incorporated by reference in their entire T cell epitope map are analyzed in order to identify alternative ties. Other techniques are known in the art. Fully human amino acid substitutions with a low risk of altering activity of antibodies can likewise be produced by various display tech the final antibody. A range of alternativeV and V, sequences nologies, e.g., phage display or other viral display systems, as are designed comprising combinations of amino acid Substi described in more detail elsewhere herein. 30 tutions and these sequences are Subsequently incorporated Sp35 antibodies, orantigen-binding fragments, variants, or into a range of binding polypeptides, e.g., Sp35-specific anti derivatives thereof of the invention can be made or manufac bodies or immunospecific fragments thereof for use in the tured using techniques that are known in the art. In certain diagnostic and treatment methods disclosed herein, which are embodiments, antibody molecules or fragments thereof are then tested for function. Typically, between 12 and 24 variant “recombinantly produced, i.e., are produced using recombi 35 antibodies are generated and tested. Complete heavy and light nant DNA technology. Exemplary techniques for making chain genes comprising modified V and human C regions are antibody molecules or fragments thereof are discussed in then cloned into expression vectors and the Subsequent plas more detail elsewhere herein. mids introduced into cell lines for the production of whole Sp35 antibodies, orantigen-binding fragments, variants, or antibody. The antibodies are then compared in appropriate derivatives thereof of the invention also include derivatives 40 biochemical and biological assays, and the optimal variant is that are modified, e.g., by the covalent attachment of any type identified. of molecule to the antibody such that covalent attachment Sp35 antibodies, orantigen-binding fragments, variants, or does not prevent the antibody from specifically binding to its derivatives thereof of the invention may be generated by any cognate epitope. For example, but not by way of limitation, suitable method known in the art. Polyclonal antibodies to an the antibody derivatives include antibodies that have been 45 antigen of interest can be produced by various procedures modified, e.g., by glycosylation, acetylation, pegylation, well known in the art. For example, an Sp35 antibody, e.g., a phosphorylation, amidation, derivatization by known protect binding polypeptide, e.g., an Sp35-specific antibody or ing/blocking groups, proteolytic cleavage, linkage to a cellu immunospecific fragment thereof can be administered to lar ligand or other protein, etc. Any of numerous chemical various host animals including, but not limited to, rabbits, modifications may be carried out by known techniques, 50 mice, rats, chickens, hamsters, goats, donkeys, etc., to induce including, but not limited to specific chemical cleavage, the production of Sera containing polyclonal antibodies spe acetylation, formylation, metabolic synthesis oftunicamycin, cific for the antigen. Various adjuvants may be used to etc. Additionally, the derivative may contain one or more increase the immunological response, depending on the host non-classical amino acids. species, and include but are not limited to, Freund's (com In certain embodiments, Sp35 antibodies, or antigen-bind 55 plete and incomplete), mineral gels such as aluminum ing fragments, variants, or derivatives thereof of the invention hydroxide, Surface active Substances Such as lysolecithin, will not elicit a deleterious immune response in the animal to pluronic polyols, polyanions, peptides, oil emulsions, key be treated, e.g., in a human. In one embodiment, Sp35 anti hole limpet hemocyanins, dinitrophenol, and potentially use bodies, or antigen-binding fragments, variants, or derivatives ful human adjuvants such as BCG (bacille Calmette-Guerin) thereof of the invention are modified to reduce their immu 60 and Corynebacterium parvum. Such adjuvants are also well nogenicity using art-recognized techniques. For example, known in the art. antibodies can be humanized, primatized, deimmunized, or Monoclonal antibodies can be prepared using a wide vari chimeric antibodies can be made. These types of antibodies ety of techniques known in the art including the use of hybri are derived from a non-human antibody, typically a murine or doma, recombinant, and phage display technologies, or a primate antibody, that retains or Substantially retains the anti 65 combination thereof. For example, monoclonal antibodies gen-binding properties of the parent antibody, but which is can be produced using hybridoma techniques including those less immunogenic in humans. This may be achieved by vari known in the art and taught, for example, in Harlow et al., US 8,551,476 B2 39 40 Antibodies: A Laboratory Manual, Cold Spring Harbor Labo procedures such as, for example, protein-A, hydroxylapatite ratory Press, 2nd ed. (1988); Hammerling et al., in: Mono chromatography, gel electrophoresis, dialysis or affinity clonal Antibodies and T-Cell Hybridomas Elsevier, N.Y., chromatography. 563-681 (1981) (said references incorporated by reference in Antibody fragments that recognize specific epitopes may their entireties). The term “monoclonal antibody” as used be generated by known techniques. For example, Fab and herein is not limited to antibodies produced through hybri F(ab')2 fragments may be produced by proteolytic cleavage of doma technology. The term “monoclonal antibody' refers to immunoglobulin molecules, using enzymes such as papain an antibody that is derived from a single clone, including any (to produce Fab fragments) or pepsin (to produce F(ab') eukaryotic, prokaryotic, or phage clone, and not the method fragments). F(ab') fragments contain the variable region, the 10 light chain constant region and the C1 domain of the heavy by which it is produced. Thus, the term “monoclonal anti chain. body' is not limited to antibodies produced through hybri Those skilled in the art will also appreciate that DNA doma technology. Monoclonal antibodies can be prepared encoding antibodies or antibody fragments (e.g., antigen using Sp35 knockout mice to increase the regions of epitope binding sites) may also be derived from antibody libraries, recognition. Monoclonal antibodies can be prepared using a 15 Such as phage display libraries. In a particular, such phage can wide variety of techniques known in the art including the use be utilized to display antigen-binding domains expressed of hybridoma and recombinant and phage display technology from a repertoire or combinatorial antibody library (e.g., as described elsewhere herein. human or murine). Phage expressing an antigen binding Using art recognized protocols, in one example, antibodies domain that binds the antigen of interest can be selected or are raised in mammals by multiple Subcutaneous or intrap identified with antigen, e.g., using labeled antigen or antigen eritoneal injections of the relevant antigen (e.g., purified bound or captured to a solid surface or bead. Phage used in tumor associated antigens Such as Sp35 or cells or cellular these methods are typically filamentous phage including fa extracts comprising Such antigens) and an adjuvant. This and M13 binding domains expressed from phage with Fab, Fv immunization typically elicits an immune response that com OE DAB (individual Fv region from light or heavy chains) or prises production of antigen-reactive antibodies from acti 25 disulfide stabilized FV antibody domains recombinantly vated splenocytes or lymphocytes. While the resulting anti fused to either the phage gene III or gene VIII protein. Exem bodies may be harvested from the serum of the animal to plary methods are set forth, for example, in EP 368 684 B1; provide polyclonal preparations, it is often desirable to isolate U.S. Pat. No. 5,969,108, Hoogenboom, H. R. and Chames, individual lymphocytes from the spleen, lymph nodes or Immunol. Today 21:371 (2000): Nagy et al. Nat. Med. 8:801 peripheral blood to provide homogenous preparations of 30 (2002); Huie et al., Proc. Natl. Acad. Sci. USA 98:2682 monoclonal antibodies (MAbs). Preferably, the lymphocytes (2001): Lui et al., J. Mol. Biol. 315:1063 (2002), each of are obtained from the spleen. which is incorporated herein by reference. Several publica In this well known process (Kohler et al., Nature 256:495 tions (e.g., Marks et al., Bio/Technology 10:779-783 (1992)) (1975)) the relatively short-lived, or mortal, lymphocytes have described the production of high affinity human anti from a mammal which has been injected with antigen are 35 bodies by chain shuffling, as well as combinatorial infection fused with an immortal tumor cell line (e.g. a myeloma cell and in vivo recombination as a strategy for constructing large line), thus, producing hybrid cells or “hybridomas' which are phage libraries. In another embodiment, Ribosomal display both immortal and capable of producing the genetically can be used to replace bacteriophage as the display platform coded antibody of the B cell. The resulting hybrids are seg (see, e.g., Hanes et al., Nat. Biotechnol. 18:1287 (2000); regated into single genetic strains by selection, dilution, and 40 Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750 (2001); or regrowth with each individual strain comprising specific Irving et al., J. Immunol. Methods 248:31 (2001)). In yet genes for the formation of a single antibody. They produce another embodiment, cell surface libraries can be screened antibodies which are homogeneous against a desired antigen for antibodies (Boder et al., Proc. Natl. Acad. Sci. USA and, in reference to their pure genetic parentage, are termed 97: 10701 (2000); Daugherty et al., J. Immunol. Methods 243: "monoclonal.” 45 211 (2000)). Such procedures provide alternatives to tradi Hybridoma cells thus prepared are seeded and grown in a tional hybridoma techniques for the isolation and Subsequent suitable culture medium that preferably contains one or more cloning of monoclonal antibodies. substances that inhibit the growth or survival of the unfused, In phage display methods, functional antibody domains are parental myeloma cells. Those skilled in the art will appreci displayed on the Surface of phage particles which carry the ate that reagents, cell lines and media for the formation, 50 polynucleotide sequences encoding them. For example, DNA selection and growth of hybridomas are commercially avail sequences encoding V and V regions are amplified from able from a number of sources and standardized protocols are animal clNA libraries (e.g., human or murine cDNA librar well established. Generally, culture medium in which the ies of lymphoid tissues) or synthetic cDNA libraries. In cer hybridoma cells are growing is assayed for production of tain embodiments, the DNA encoding the V, and V, regions monoclonal antibodies against the desired antigen. Prefer 55 are joined together by an schv linker by PCR and cloned into ably, the binding specificity of the monoclonal antibodies a phagemid vector (e.g., p CANTAB 6 orpComb3 HSS). The produced by hybridoma cells is determined by in vitro assays vector is electroporated in E. coli and the E. coli is infected Such as immunoprecipitation, radioimmunoassay (RIA) or with helper phage. Phage used in these methods are typically enzyme-linked immunoabsorbent assay (ELISA). After filamentous phage including fl and M13 and the V or V. hybridoma cells are identified that produce antibodies of the 60 regions are usually recombinantly fused to either the phage desired specificity, affinity and/or activity, the clones may be gene III or gene VIII. Phage expressing an antigen binding Subcloned by limiting dilution procedures and grown by stan domain that binds to an antigen of interest (i.e., an Sp35 dard methods (Goding, Monoclonal Antibodies: Principles polypeptide or a fragment thereof) can be selected or identi and Practice, Academic Press, pp 59-103 (1986)). It will fied with antigen, e.g., using labeled antigen orantigenbound further be appreciated that the monoclonal antibodies 65 or captured to a solid Surface or bead. secreted by the subclones may be separated from culture Additional examples of phage display methods that can be medium, ascites fluid or serum by conventional purification used to make the antibodies include those disclosed in Brink US 8,551,476 B2 41 42 man et al., J. Immunol. Methods 182:41-50 (1995); Ames et Completely human antibodies are particularly desirable for al., J. Immunol. Methods 184:177-186 (1995); Kettleborough therapeutic treatment of human patients. Human antibodies et al., Eur: J. Immunol. 24:952-958 (1994); Persic et al., Gene can be made by a variety of methods known in the art includ 187:9-18 (1997); Burton et al., Advances in Immunology ing phage display methods described above using antibody 57:191-280 (1994); PCT Application No. PCT/GB91/01134; libraries derived from human immunoglobulin sequences. PCT publications WO 90/02809; WO 91/10737; WO See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT 92/01047; WO92/18619; WO 93/11236: WO95/15982; WO publications WO 98/46645, WO 98/50433, WO 98/24893, 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403, WO 98/16654, WO 96/34096, WO 96/33735, and WO 484: 5,580,717:5,427,908; 5,750,753; 5,821,047; 5,571,698; 91/10741; each of which is incorporated herein by reference 5,427,908: 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 10 in its entirety. 5.969,108; each of which is incorporated herein by reference Human antibodies can also be produced using transgenic in its entirety. mice which are incapable of expressing functional endog As described in the above references, after phage selection, enous immunoglobulins, but which can express human the antibody coding regions from the phage can be isolated immunoglobulin genes. For example, the human heavy and and used to generate whole antibodies, including human anti 15 light chain immunoglobulin gene complexes may be intro bodies, or any other desired antigen binding fragment, and duced randomly or by homologous recombination into mouse expressed in any desired host, including mammalian cells, embryonic stem cells. Alternatively, the human variable insect cells, plant cells, yeast, and bacteria. For example, region, constant region, and diversity region may be intro techniques to recombinantly produce Fab, Fab' and F(ab') duced into mouse embryonic stem cells in addition to the fragments can also be employed using methods known in the human heavy and light chain genes. The mouse heavy and art such as those disclosed in PCT publication WO92/22324; light chain immunoglobulin genes may be rendered non Mullinax et al., BioTechniques 12(6):864-869 (1992); and functional separately or simultaneously with the introduction Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science of human immunoglobulin loci by homologous recombina 240: 1041-1043 (1988) (said references incorporated by ref tion. In particular, homozygous deletion of the JH region erence in their entireties). 25 prevents endogenous antibody production. The modified Examples of techniques which can be used to produce embryonic stem cells are expanded and microinjected into single-chain Fvs and antibodies include those described in blastocysts to produce chimeric mice. The chimeric mice are U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Meth then bred to produce homozygous offspring that express ods in Enzymology 203:46-88 (1991); Shu et al., PNAS human antibodies. The transgenic mice are immunized in the 90:7995–7999 (1993); and Skerra et al., Science 240:1038 30 normal fashion with a selected antigen, e.g., all or a portion of 1040 (1988). For some uses, including in vivo use of antibod a desired target polypeptide. Monoclonal antibodies directed ies in humans and in vitro detection assays, it may be prefer against the antigen can be obtained from the immunized, able to use chimeric, humanized, or human antibodies. A transgenic mice using conventional hybridoma technology. chimericantibody is a molecule in which different portions of The human immunoglobulin transgenes harbored by the the antibody are derived from different animal species, such 35 transgenic mice rearrange during B-cell differentiation, and as antibodies having a variable region derived from a murine Subsequently undergo class Switching and somatic mutation. monoclonal antibody and a human immunoglobulin constant Thus, using Such a technique, it is possible to produce thera region. Methods for producing chimeric antibodies are peutically useful IgG, IgA, IgM and IgE antibodies. For an known in the art. See, e.g., Morrison, Science 229:1202 overview of this technology for producing human antibodies, (1985): Oiet al., BioTechniques 4:214 (1986); Gillies et al., J. 40 see Lonberg and Huszar Int. Rev. Immunol. 13:65-93 (1995). Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. For a detailed discussion of this technology for producing 5,807,715; 4,816,567; and 4,816397, which are incorporated human antibodies and human monoclonal antibodies and pro herein by reference in their entireties. Humanized antibodies tocols for producing Such antibodies, see, e.g., PCT publica are antibody molecules derived from a non-human species tions WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. antibody that bind the desired antigen having one or more 45 Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016: complementarity determining regions (CDRS) from the non 5,545,806:5,814,318; and 5,939,598, which are incorporated human species and framework regions from a human immu by reference herein in their entirety. In addition, companies noglobulin molecule. Often, framework residues in the such as Abgenix, Inc. (Freemont, Calif.) and GenPharm (San human framework regions will be substituted with the corre Jose, Calif.) can be engaged to provide human antibodies sponding residue from the CDR donorantibody to alter, pref 50 directed against a selected antigen using technology similar erably improve, antigen binding. These framework Substitu to that described above. tions are identified by methods well known in the art, e.g., by Completely human antibodies which recognize a selected modeling of the interactions of the CDR and framework resi epitope can be generated using a technique referred to as dues to identify framework residues important for antigen 'guided selection.” In this approach a selected non-human binding and sequence comparison to identify unusual frame 55 monoclonal antibody, e.g., a mouse antibody, is used to guide work residues at particular positions. (See, e.g., Queen et al., the selection of a completely human antibody recognizing the U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 same epitope. (Jespers et al., Bio/Technology 12:899-903 (1988), which are incorporated herein by reference in their (1988). See also, U.S. Pat. No. 5,565,332, which is incorpo entireties.) Antibodies can be humanized using a variety of rated by reference in its entirety.) techniques known in the art including, for example, CDR 60 Further, antibodies to target polypeptides of the invention grafting (EP239,400; PCT publication WO 91/09967; U.S. can, in turn, be utilized to generate anti-idiotype antibodies Pat. Nos. 5.225,539; 5,530,101; and 5,585,089), veneering or that "mimic target polypeptides using techniques well resurfacing (EP 592,106; EP 519,596: Padlan, Molecular known to those skilled in the art. (See, e.g., Greenspan & Immunology 28(4/5):489-498 (1991); Studnicka et al., Pro Bona, FASEB.J. 7(5):437-444 (1989) and Nissinoff, J. Immu tein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 65 mol. 147(8):2429-2438 (1991)). For example, antibodies 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565, which bind to and competitively inhibit polypeptide multim 332, which is incorporated by reference in its entirety). erization and/or binding of a polypeptide of the invention to a US 8,551,476 B2 43 44 ligand can be used to generate anti-idiotypes that "mimic” the tions improve binding of the antibody to its antigen. Addi polypeptide multimerization and/or binding domain and, as a tionally, Such methods may be used to make amino acid consequence, bind to and neutralize polypeptide and/or its Substitutions or deletions of one or more variable region cys ligand. Such neutralizing anti-idiotypes or Fab fragments of teine residues participating in an intrachain disulfide bond to Such anti-idiotypes can be used in therapeutic regimens to 5 generate antibody molecules lacking one or more intrachain neutralize polypeptide ligand. For example, Such anti-idio disulfide bonds. Other alterations to the polynucleotide are typic antibodies can be used to binda desired target polypep encompassed by the present invention and within the skill of tide and/or to bind its ligands/receptors, and thereby block its the art. biological activity. In addition, techniques developed for the production of In another embodiment, DNA encoding desired mono 10 "chimericantibodies' (Morrison et al., Proc. Natl. Acad. Sci. clonal antibodies may be readily isolated and sequenced 81:851-855 (1984); Neuberger et al., Nature 312:604-608 using conventional procedures (e.g., by using oligonucleotide (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing probes that are capable of binding specifically to genes genes from a mouse antibody molecule of appropriate antigen encoding the heavy and light chains of murine antibodies). specificity together with genes from a human antibody mol The isolated and subcloned hybridoma cells serve as a pre 15 ecule of appropriate biological activity can be used. As used ferred source of such DNA. Once isolated, the DNA may be herein, a chimeric antibody is a molecule in which different placed into expression vectors, which are then transfected portions are derived from different animal species, such as into prokaryotic or eukaryotic host cells Such as E. coli cells, those having a variable region derived from a murine mono simian COS cells, Chinese Hamster Ovary (CHO) cells or clonal antibody and a human immunoglobulin constant myeloma cells that do not otherwise produce immunoglobu region, e.g., humanized antibodies. lins. More particularly, the isolated DNA (which may be Alternatively, techniques described for the production of synthetic as described herein) may be used to clone constant single chain antibodies (U.S. Pat. No. 4,694,778; Bird, Sci and variable region sequences for the manufacture antibodies ence 242:423-442 (1988); Hustonet al., Proc. Natl. Acad. Sci. as described in Newman et al., U.S. Pat. No. 5,658,570, filed USA 85:5879-5883 (1988); and Ward et al., Nature 334:544 Jan. 25, 1995, which is incorporated by reference herein. 25 554 (1989)) can be adapted to produce single chain antibod Essentially, this entails extraction of RNA from the selected ies. Single chain antibodies are formed by linking the heavy cells, conversion to cDNA, and amplification by PCR using Ig and light chain fragments of the FV region via an amino acid specific primers. Suitable primers for this purpose are also bridge, resulting in a single chain antibody. Techniques for described in U.S. Pat. No. 5,658,570. As will be discussed in the assembly of functional Fv fragments in E. coli may also be more detail below, transformed cells expressing the desired 30 used (Skerra et al., Science 242:1038-1041 (1988)). antibody may be grown up in relatively large quantities to Yet other embodiments of the present invention comprise provide clinical and commercial supplies of the immunoglo the generation of human or substantially human antibodies in bulin. transgenic animals (e.g., mice) that are incapable of endog In one embodiment, an Sp35 antibody of the invention enous immunoglobulin production (see e.g., U.S. Pat. Nos. comprises at least one heavy or light chain CDR of an anti 35 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of body molecule. In another embodiment, an Sp35 antibody of which is incorporated herein by reference). For example, it the invention comprises at least two CDRs from one or more has been described that the homozygous deletion of the anti antibody molecules. In another embodiment, an Sp35 anti body heavy-chain joining region in chimeric and germ-line body of the invention comprises at least three CDRs from one mutant mice results in complete inhibition of endogenous or more antibody molecules. In another embodiment, an Sp35 40 antibody production. Transfer of a human immunoglobulin antibody of the invention comprises at least four CDRs from gene array to such germ line mutant mice will result in the one or more antibody molecules. In another embodiment, an production of human antibodies upon antigen challenge. Sp35 antibody of the invention comprises at least five CDRs Another preferred means of generating human antibodies from one or more antibody molecules. In another embodi using SCID mice is disclosed in U.S. Pat. No. 5,811,524 ment, an Sp35 antibody of the invention comprises at least six 45 which is incorporated herein by reference. It will be appreci CDRs from one or more antibody molecules. Exemplary ated that the genetic material associated with these human antibody molecules comprising at least one CDR that can be antibodies may also be isolated and manipulated as described included in the subject Sp35 antibodies are described herein. herein. In a specific embodiment, the amino acid sequence of the Yet another highly efficient means for generating recom heavy and/or light chain variable domains may be inspected 50 binant antibodies is disclosed by Newman, Biotechnology 10: to identify the sequences of the complementarity determining 1455-1460 (1992). Specifically, this technique results in the regions (CDRs) by methods that are well know in the art, e.g., generation of primatized antibodies that contain monkey vari by comparison to known amino acid sequences of other heavy able domains and human constant sequences. This reference and light chain variable regions to determine the regions of is incorporated by reference in its entirety herein. Moreover, sequence hypervariability. Using routine recombinant DNA 55 this technique is also described in commonly assigned U.S. techniques, one or more of the CDRs may be inserted within Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which framework regions, e.g., into human framework regions to is incorporated herein by reference. humanize a non-human antibody. The framework regions In another embodiment, lymphocytes can be selected by may be naturally occurring or consensus framework regions, micromanipulation and the variable genes isolated. For and preferably human framework regions (see, e.g., Chothia 60 example, peripheral blood mononuclear cells can be isolated et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human from an immunized mammal and cultured for about 7 days in framework regions). Preferably, the polynucleotide gener vitro. The cultures can be screened for specific IgGs that meet ated by the combination of the framework regions and CDRs the screening criteria. Cells from positive wells can be iso encodes an antibody that specifically binds to at least one lated. Individual Ig-producing B cells can be isolated by epitope of a desired polypeptide, e.g., Sp35. Preferably, one 65 FACS or by identifying them in a complement-mediated or more amino acid Substitutions may be made within the hemolytic plaque assay. Ig-producing B cells can be micro framework regions, and, preferably, the amino acid substitu manipulated into a tube and the V and V, genes can be US 8,551,476 B2 45 46 amplified using, e.g., RT-PCR. The V and V, genes can be to enhance desirable characteristics such as effector function cloned into an antibody expression vector and transfected into or provide for more cytotoxin or carbohydrate attachment. In cells (e.g., eukaryotic or prokaryotic cells) for expression. such embodiments it may be desirable to insert or replicate Alternatively, antibody-producing cell lines may be specific sequences derived from selected constant region selected and cultured using techniques well known to the 5 domains. skilled artisan. Such techniques are described in a variety of The present invention also provides antibodies that com laboratory manuals and primary publications. In this respect, prise, consist essentially of, or consist of variants (including techniques suitable for use in the invention as described derivatives) of antibody molecules (e.g., the V regions and/ below are described in Current Protocols in Immunology, or V regions) described herein, which antibodies or frag Coligan et al., Eds. Green Publishing Associates and Wiley 10 ments thereof immunospecifically bind to an Sp35 polypep Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including tide or fragment or variant thereof. Standard techniques Supplements. known to those of skill in the art can be used to introduce Antibodies for use in the diagnostic and therapeutic meth mutations in the nucleotide sequence encoding an Sp35 anti ods disclosed herein can be produced by any method known 15 body, including, but not limited to, site-directed mutagenesis in the art for the synthesis of antibodies, in particular, by and PCR-mediated mutagenesis which result in amino acid chemical synthesis or preferably, by recombinant expression substitutions. Preferably, the variants (including derivatives) techniques as described herein. encode less than 50 amino acid substitutions, less than 40 In one embodiment, an Sp35 antibody, or antigen-binding amino acid Substitutions, less than 30 amino acid substitu fragment, variant, or derivative thereof of the invention com tions, less than 25 amino acid substitutions, less than 20 prises a synthetic constant region wherein one or more amino acid Substitutions, less than 15 amino acid substitu domains are partially or entirely deleted (“domain-deleted tions, less than 10amino acid Substitutions, less than 5 amino antibodies’). In certain embodiments compatible modified acid Substitutions, less than 4 amino acid Substitutions, less antibodies will comprise domain deleted constructs or vari than 3 amino acid Substitutions, or less than 2 amino acid ants wherein the entire C2 domain has been removed (AC2 25 substitutions relative to the reference V, region, VCDR1, constructs). For other embodiments a short connecting pep VCDR2, VCDR3, V, region, V, CDR1, VCDR2, or tide may be substituted for the deleted domain to provide V, CDR3. A "conservative amino acid substitution' is one in flexibility and freedom of movement for the variable region. which the amino acid residue is replaced with an amino acid Those skilled in the art will appreciate that such constructs are residue having a side chain with a similar charge. Families of particularly preferred due to the regulatory properties of the 30 amino acid residues having side chains with similar charges C2 domain on the catabolic rate of the antibody. Domain have been defined in the art. These families include amino deleted constructs can be derived using a vector (e.g., from acids with basic side chains (e.g., lysine, arginine, histidine), Biogen IDEC Incorporated) encoding an IgG human con acidic side chains (e.g., aspartic acid, glutamic acid), stant domain (see, e.g., WO 02/060955A2 and WO02/ uncharged polar side chains (e.g., glycine, asparagine, 096948A2, which are incorporated by reference in their 35 entireties). This exemplary vector was engineered to delete glutamine, serine, threonine, tyrosine, cysteine), nonpolar the C2 domain and provide a synthetic vector expressing a side chains (e.g., alanine, Valine, leucine, isoleucine, proline, domain deleted IgG constant region. phenylalanine, methionine, tryptophan), beta-branched side In certain embodiments, Sp35 antibodies, or antigen-bind chains (e.g., threonine, Valine, isoleucine) and aromatic side ing fragments, variants, or derivatives thereof of the invention 40 chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). are minibodies. Minibodies can be made using methods Alternatively, mutations can be introduced randomly along described in the art (see, e.g., see e.g., U.S. Pat. No. 5,837.821 all or part of the coding sequence, such as by Saturation or WO94/09817A1, which are incorporated by reference in mutagenesis, and the resultant mutants can be screened for their entireties). biological activity to identify mutants that retain activity In one embodiment, an Sp35 antibody, or antigen-binding 45 (e.g., the ability to bind an Sp35 polypeptide). fragment, variant, or derivative thereof of the invention com For example, it is possible to introduce mutations only in prises an immunoglobulin heavy chain having deletion or framework regions or only in CDR regions of an antibody Substitution of a few or even a single amino acid as long as it molecule. Introduced mutations may be silent or neutral mis permits association between the monomeric Subunits. For sense mutations, i.e., have no, or little, effect on an antibody’s example, the mutation of a singleamino acid in selected areas 50 ability to bind antigen. These types of mutations may be of the C2 domain may be enough to Substantially reduce Fc useful to optimize codon usage, or improve a hybridoma's binding and thereby increase tumor localization. Similarly, it antibody production. Alternatively, non-neutral missense may be desirable to simply delete that part of one or more mutations may alteran antibody's ability to bind antigen. The constant region domains that control the effector function location of most silent and neutral missense mutations is (e.g. complement binding) to be modulated. Such partial 55 likely to be in the framework regions, while the location of deletions of the constant regions may improve selected char most non-neutral missense mutations is likely to be in CDR, acteristics of the antibody (serum half-life) while leaving though this is not an absolute requirement. One of skill in the other desirable functions associated with the subject constant art would be able to design and test mutant molecules with region domain intact. Moreover, as alluded to above, the desired properties Such as no alteration in antigen binding constant regions of the disclosed antibodies may be synthetic 60 activity or alteration in binding activity (e.g., improvements through the mutation or Substitution of one or more amino in antigenbinding activity or change in antibody specificity). acids that enhances the profile of the resulting construct. In Following mutagenesis, the encoded protein may routinely be this respect it may be possible to disrupt the activity provided expressed and the functional and/or biological activity of the by a conserved binding site (e.g. Fc binding) while Substan encoded protein, (e.g., ability to immunospecifically bind at tially maintaining the configuration and immunogenic profile 65 least one epitope of an Sp35 polypeptide) can be determined of the modified antibody. Yet other embodiments comprise using techniques described herein or by routinely modifying the addition of one or more amino acids to the constant region techniques known in the art.

US 8,551,476 B2 51 52 TABLE 4 - Continued Reference VH CDR1, CDR2, and CDR3 amino acid sequences Antibody Name WH-CDR1 WH-CDR2 WH-CDR3

L1a. 12 P = GDSWSDNSAAWG P = RIYYRSKWYNDYAWSWKS P GRHEYGGLGYAEAMDH (SEQ ID NO: 228) (SEQ ID NO: 229) (SEQ ID NO: 230)

L1a. 13 P = GFTFSSYAMS P = AISGSGGSTYYADSWKG P HYTYMHEEDY (SEQ ID NO: 231) (SEQ ID NO: 232) (SEQ ID NO: 233) Determined by the Kabat System (see Supra). N = nucleotide sequence, P = polypeptide sequence.

In certain embodiments, an antibody orantigen-binding frag 15 28-D02 (Li10), 30-B01 (Li11), 34-B03 (L12), Li13, Li32, ment comprising the VH encoded by the polynucleotide spe Li33, Li34, 3383 (L1a.1), 3495 (L1 a.2), 3563 (L1 a.3), 3564 cifically or preferentially binds to Sp35. (L1 a.4), 3565 (L1 a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 In another embodiment, the present invention provides an (L1a.8), 3569 (L1a.9), 3570 (L1a. 10), 3571 (L1a. 11), 3582 isolated polynucleotide comprising, consisting essentially of (L1a. 12), and 1968 (L1 a.13), or will competitively inhibit such a monoclonal antibody from binding to Sp35. or consisting of a nucleic acid encoding an immunoglobulin In certain embodiments, an antibody or antigen-binding heavy chain variable region (VH) in which the CDR1, CDR2, fragment thereof comprising, consisting essentially of or and CDR3 regions have polypeptide sequences which are consisting of a VH encoded by one or more of the polynucle identical to the CDR1, CDR2, and CDR3 groups shown in otides described above specifically or preferentially binds to Table 4. In certain embodiments, an antibody or antigen an Sp35 polypeptide or fragment thereof, or a Sp35 variant binding fragment comprising the VH encoded by the poly 25 polypeptide, with an affinity characterized by a dissociation nucleotide specifically or preferentially binds to Sp35. constant (Ki) no greater than 5x10° M, 10° M, 5x10 M, In a further aspect, the present invention provides an iso 10 M, 5x10* M, 10 M, 5x105 M, 10-s M, 5x106 M, lated polynucleotide comprising, consisting essentially of or 10 M, 5x107 M, 107 M., 5x10 M, 10 M, 5x10 M, consisting of a nucleic acid encoding an immunoglobulin 10- M., 5x100 M, 100M, 5x10 M, 10 M, 5x10-12 heavy chain variable region (VH) in which the CDR1, CDR2, 30 M, 10° M, 5x10' M, 10 M, 5x10 M, 10 M, and CDR3 regions are encoded by nucleotide sequences 5x10's M, or 10 M. which are identical to the nucleotide sequences which encode In another embodiment, the present invention provides an the CDR1, CDR2, and CDR3 groups shown in Table 4. In isolated polynucleotide comprising, consisting essentially of certain embodiments, an antibody or antigen-binding frag or consisting of a nucleic acid encoding an immunoglobulin ment comprising the VH encoded by the polynucleotide spe 35 light chain variable region (VL), where at least one of the cifically or preferentially binds to Sp35. CDRs of the light chain variable region or at least two of the In certain embodiments, an antibody or antigen-binding CDRs of the light chain variable region are at least 80%, 85%, fragment thereof comprising, consisting essentially of, or 90% or 95% identical to reference light chain CDR1, CDR2, consisting of a VH encoded by one or more of the polynucle or CDR3 amino acid sequences from monoclonal Sp35 anti otides described above specifically or preferentially binds to 40 bodies disclosed herein. Alternatively, the CDR1, CDR2, and the same epitope as a monoclonal antibody selected from the CDR3 regions of the VL are at least 80%, 85%, 90% or 95% group consisting of 201", 3A3, 3A6, 1A7, 1G7. 2B10, 2C11. identical to reference light chain CDR1, CDR2, and CDR3 2F3, 3P1D10.2C3, 3P1E1 1.3B7, 3P2C6.3G10.2H7, amino acid sequences from monoclonal Sp35 antibodies dis 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, closed herein. Thus, according to this embodiment a light 3P4C5.1 D8, 3P4C8.2G9, 30-C12 (L100), 38-D01 (LiO22), 45 chain variable region of the invention has CDR1, CDR2, or 35-E04 (LiO33), 36-C09 (LiO4), 30-A11 (LiO5), 34-F02 CDR3 polypeptide sequences related to the polypeptide (LiO6), 29-E07 (LiO7), 34-G04 (LiO8), 36-A12 (LiO9), sequences shown in Table 5: TABLE 5

Reference WL CDR1, CDR2, and CDR3 amino acid sequences* Antibody Name WL-CDR1 WL-CDR2 WL-CDR3

Li10 P RASQGIGNWLA P = AASSLES P = OOAQTFPLT (SEO ID NO : 87) (SEO ID NO: 89) (SEQ ID NO: 91 N CGGGCGAGTCAGGG N = GCTGCATCCAGTTTGGAAAGT N = CAACAGGCTCAGAC TATTGGCAACTGGTTAGCC (SEQ ID NO: 88) TTTCCCGCT CACC (SEQ ID NO: 86) (SEO ID NO: 9 O)

LiO7 P = SGDOLGDKHWA P = LDIKRPA P = OAWDIKTV (SEO ID NO: 93) (SEO ID NO: 95) (SEO ID NO: 97 N TCTGGAGATCAGTTG N = CTAGACATTAAGAGGCCCGCA N = CAGGCGTGGGACATC GGTGACAAACATGTGGCT (SEQ ID NO: 94) AAGACGGTC (SEQ ID NO: 92) (SEQ ID NO: 96)

US 8,551,476 B2 55 56 TABLE 5- continued Reference VL CDR1, CDR2, and CDR3 amino acid sequences Antibody Name WL-CDR1 WL-CDR2

1a. O2 P = RASOSITNSYLG P = DASSRAT d QQASDAPE (SEO ID NO. 237) (SEQ ID NO: 238) (S e Q ID NO: 239)

1a. O3 P = RASOGINFWLN P = AGSNLOS d MODSDFPF (SEQ ID NO: 240) (SEQ ID NO: 241) (S e Q ID NO: 242)

1a. O4 P = TGSSSNIGAGYDWS P = RNNNRPS P = OTYDNSTD (SEQ ID NO: 243) (SEQ ID NO: 244) (SEQ ID NO: 245)

1a. Os P = SGDNIRSYYWH P = EDSNRPS d QSYDSAILLH (SEQ ID NO: 246) (SEO ID NO: 247) (SEQS e ID NO: 248)

1a. O6 P = RSSOSLVLRTGYTYLN P = LVSNRAS d (SEQ ID NO: 249) (SEQ ID NO: 25O) (SEQS e ID NO: 251)

1a. O7 P = RASOSWSYOYLA P = GASSRAT d OQYGSVPR (SEQ ID NO: 252) (SEQ ID NO: 253) (SEQS e ID NO: 254)

1a. O8 P = SGDSLGSYYWH P = DDNDRPS P = SAYDYSART (SEO ID NO: 255) (SEQ ID NO: 256) (SEO ID NO: 257)

1a. O9 P = SGDNLGSKYWS P = DDDDRPS d SSYDFLNIGL (SEO ID NO: 258) (SEO ID NO: 259) ( SEQe D NO: 260)

1a. 10 P = SGDSLGKKSWH P = EDSERPS d (SEQ ID NO: 261) (SEQ ID NO: 262) (SEQS e ID NO: 263)

1a. 11 P = SGDNLGKKYWG P = DDDNRPS OSYDDTSI (SEQ ID NO: 264) (SEQ ID NO: 265) D NO: 266)

1a. 12 P = SGDSLGNKYWH P = DDSDRPS QTWDYVGY (SEO ID NO: 267) (SEQ ID NO: 268) D NO: 269)

1a. 13 P = TGTSSDWGGYNYWS P = DWSNRPS = QSYDRYRLKN (SEO ID NO: 27 O) (SEO ID NO: 271) D NO: 272) Determined by the Kabat System (see Supra). N = nucleotide sequence, P = polypeptide sequence.

In certain embodiments, an antibody orantigen-binding frag 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, ment comprising the VL encoded by the polynucleotide spe 40 3P4C5.1 D8, 3P4C8.2G9, 30-C12 (L100), 38-D01 (LiO2), cifically or preferentially binds to Sp35. 35-E04 (LiO3), 36-C09 (LiO4), 30-A11 (LiO5), 34-F02 In another embodiment, the present invention provides an (LiO6), 29-E07 (LiO7), 34-G04 (LiO8), 36-A12 (LiO9), isolated polynucleotide comprising, consisting essentially of 28-D02 (Li10), 30-B01 (Li11), 34-B03 (Li12), Li13, Li32, or consisting of a nucleic acid encoding an immunoglobulin Li33, Li34, 3383 (L1a.1), 3495 (L1 a.2), 3563 (L1 a.3), 3564 light chain variable region (VL) in which the CDR1, CDR2, 45 (L1 a.4), 3565 (L1 a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 and CDR3 regions have polypeptide sequences which are (L1a.8), 3569 (L1a.9), 3570 (L1a. 10), 3571 (L1a. 11), 3582 identical to the CDR1, CDR2, and CDR3 groups shown in (L1a. 12), and 1968 (L1 a.13), or will competitively inhibit Table 5. In certain embodiments, an antibody or antigen such a monoclonal antibody from binding to Sp35. binding fragment comprising the VL encoded by the poly In certain embodiments, an antibody or antigen-binding nucleotide specifically or preferentially binds to Sp35. 50 fragment thereof comprising, consisting essentially of or In a further aspect, the present invention provides an iso consisting of a VL encoded by one or more of the polynucle lated polynucleotide comprising, consisting essentially of or otides described above specifically or preferentially binds to consisting of a nucleic acid encoding an immunoglobulin an Sp35 polypeptide or fragment thereof, or a Sp35 variant light chain variable region (VL) in which the CDR1, CDR2, polypeptide, with an affinity characterized by a dissociation and CDR3 regions are encoded by nucleotide sequences 55 constant (Ki) no greater than 5x10° M, 10° M, 5x10 M, which are identical to the nucleotide sequences which encode 10 M, 5x10* M, 10 M, 5x105 M, 10-s M, 5x106 M, the CDR1, CDR2, and CDR3 groups shown in Table 5. In 10M,5x107M, 107M,5x10M, 10M,5x10M, 10 certain embodiments, an antibody or antigen-binding frag M, 5x100 M, 100 M, 5x10' M, 10 M, 5x10'? M, ment comprising the VL encoded by the polynucleotide spe 10-12 M,5x10-13M, 10-13M, 5x10-14M, 10-14M,5x10-15 cifically or preferentially binds to Sp35. 60 M, or 10 M. In certain embodiments, an antibody or antigen-binding In a further embodiment, the present invention includes an fragment thereof comprising, consisting essentially of, or isolated polynucleotide comprising, consisting essentially of consisting of a VL encoded by one or more of the polynucle or consisting of a nucleic acid encoding a VH at least 80%, otides described above specifically or preferentially binds to 85%, 90% or 95% identical to a reference VH polypeptide the same epitope as a monoclonal antibody selected from the 65 sequence selected from the group consisting of SEQID NOs: group consisting of 201'. 3A3, 3A6, 1A7, 1G7. 2B10, 2C11. 158 to 172, 372, 376, 380, and 384 shown in Table 6. In 2F3, 3P1D10.2C3, 3P1E1 1.3B7, 3P2C6.3G10.2H7, certain embodiments, an antibody or antigen-binding frag

US 8,551,476 B2 59 60 TABLE 6-continued VH Polypeptide Sequences

SEQ ID WH Sequence NO :

Li32 EVOLLESGGGLVOPGGSLRLSCAASGFTFSAYMMOWVROAPGKGLEWVSSIS 376 PSGGNTKYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARGDYGY WFDPWGOGTLVTVSS

Li33 EVOLLESGGGLVOPGGSLRLSCAASGFTFSIYPMFWWROAPGKGLEWWSWIGP 380 SGGITKYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTATYYCAREGHNDWY FDLWGRGTLWTWSS

Li34 EVOLLESGGGLVOPGGSLRLSCAASGFTFSNYEMYWWROAPGKGLEWWSGIY 384 SSGGITVYADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCARAAILDW

In a further embodiment, the present invention includes an 29-E07 (LiO7), 34-G04 (LiO8), 36-A12 (LiO9), 28-D02 isolated polynucleotide comprising, consisting essentially of (Li10), 30-B01 (Li11), 34-B03 (Li12), Li13, Li32, Li33, or consisting of a nucleic acid encoding a VH at least 80%, Li34, 3383 (L1a. 1), 3495 (L1 a.2), 3563 (L1 a.3), 3564 85%, 90% or 95% identical to a reference VH polypeptide (L1 a.4), 3565 (L1 a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 sequence selected from the group consisting of SEQID NOs: (L1a.8), 3569 (L1a.9), 3570 (L1a. 10), 3571 (L1a. 11), 3582 158-172,372,376, 380, and 384. In certain embodiments, an (L1a. 12), and 1968 (L1 a.13), or will competitively inhibit antibody or antigen-binding fragment comprising the VH 25 such a monoclonal antibody from binding to Sp35. encoded by the polynucleotide specifically or preferentially In certain embodiments, an antibody or antigen-binding binds to Sp35. fragment thereof comprising, consisting essentially of or In another aspect, the present invention includes an isolated consisting of a VH encoded by one or more of the polynucle polynucleotide comprising, consisting essentially of, or con otides described above specifically or preferentially binds to sisting of a nucleic acid sequence encoding a VH of the 30 an Sp35 polypeptide or fragment thereof, or a Sp35 variant invention, selected from the group consisting of SEQIDNOs: polypeptide, with an affinity characterized by a dissociation 158-172,372,376, 380, and 384. In certain embodiments, an constant (Ki) no greater than 5x10° M, 10° M, 5x10 M, antibody or antigen-binding fragment comprising the VH 10 M, 5x10* M, 10 M, 5x105 M, 10-s M, 5x106 M, encoded by the polynucleotide specifically or preferentially 10 M, 5x107 M, 107 M., 5x10 M, 10 M, 5x10 M, binds to Sp35. 35 10 M,5x100M, 100M, 5x- M., 10 M, 5x10-12 M, In certain embodiments, an antibody or antigen-binding 1012 M,5x10-13M, 10-13M, 5x10M, 10-M,5x10-15 fragment thereof comprising, consisting essentially of, or M, or 10 M. consisting of a VH encoded by one or more of the polynucle In additional embodiments, the present invention includes otides described above specifically or preferentially binds to an isolated polynucleotide which encodes a heavy chain vari the same epitope as a monoclonal antibody selected from the 40 able region (V), where the polynucleotide comprises a V group consisting of (201") 3A3, 3A6, 1A7, 1G7. 2B10, 2C11. nucleic acid sequence selected from the group consisting of 2F3, 3P1D10.2C3, 3P1E1 1.3B7, 3P2C6.3G10.2H7, SEQID NOs 173 to 184,370,374,378 and 382, as shown in 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, Table 7. In certain embodiments, an antibody or antigen 3P4C5.1 D8, 3P4C8.2G9, 30-C12 (L100), 38-D01 (LiO2), binding fragment comprising the VH encoded by the poly 35-E04 (LiO3), 36-C09 (LiO4), 30-All (LiO5),34-F02 (LiO6), nucleotide specifically or preferentially binds to Sp35. TABLE F VH Polynucleotide Sequences

SEO ID WH Sequence NO :

LiO2 GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 173 GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTACTTACGAGATGATTTGGGT TCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCGGTCCTTCTGGT GGCCTTACTTGGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACA ACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACCGC CATGTATTACTGTGTACGGATTGATGATAGTAGTGGTTGGGCTTTTGATATCTGG GGCCAAGGGACCACGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCT TCCCGCTAGCACCC

LiOS GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTAC 174 GTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTATGTACTCTATGGTTTGGGT TCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATATCTCTCCTTCTGGT GGCAAGACTATGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACA ACTCTAAGAATACTTTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGC CGTGTATTACTGTGCGAGAGATTCGAGACGCCGGTATTACGATTTTTGGAGTGGT TATCACAACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCA CCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCACCC

US 8,551,476 B2 65 66 In a further embodiment, the present invention includes an In another aspect, the present invention includes an isolated isolated polynucleotide comprising, consisting essentially of polynucleotide comprising, consisting essentially of, or con or consisting of a nucleic acid encoding a VL at least 80%, sisting of a nucleic acid sequence encoding a VL having a 85%, 90% or 95% identical to a reference VL polypeptide sequence selected from the group consisting of SEQID NOs: polypeptide sequence selected from the group consisting of 273 to 286, 373, 377, 381 and 385, shown in Table 8. In SEQID NOS: 273 to 286, 373, 377, 381 and 385, shown in certain embodiments, an antibody or antigen-binding frag Table 8. In certain embodiments, an antibody or antigen ment comprising the VL encoded by the polynucleotide spe binding fragment comprising the VL encoded by the poly cifically or preferentially binds to Sp35. nucleotide specifically or preferentially binds to Sp35. TAB Ll E 8 VL Polypeptide Sequences

SEQ ID WL Sequence NO : iO2 FYSHSAOYELTOP PSVSVSPGOTASI TCSGDKLG DKFASWYOOKAGOS 273 IFODRKRLSGIPE RFSGSNSGNTATL TISGTOAM DEADYYCOAWDTNTVWFG GGTKLTV LGOPKAAP

LiO9 FYSHSAODIOMTOSPSSLSAFVGDRVAIT CRASOSIDTYLNWYOOKPG 274 LIYAAS KLEDGW PSRFSGSGTGTDF TLTIRSLO PEDFGTYYCOOSYS PPLT GGTKWE KRTWAA d

LiO6 FYSHSAODIOMTOSPSTLSASVGDRVTITCRASOSISSWLAWYOOKPG KAPN LIYAASSLRTGW PSRFRGSGSGTDF TLTISSLQ PEDFATYYCLODYSYP T OGTKLEIKRTVAA d

LiO5 FYSHSAOSVLTOP PSVSWAPGOTARISCGGDNIGSKSVHWYOORPGOA 276 WYDDYDRPSGIPE RFSGSNSGDTAIL TITRVEVG DEADFYCOVRDS RTEERW GGGTKVTVLGOPKAAP

LiO8 FYSHSAODIOMTOS PSSLSASVGDRVTITCOASO DISYYLNWYOOKPG 277 WLIYDVSNLOTGV PSRFSGSASATDF SSLOPE DIATYYCOOSDN PLT GGTKWEIKRTWAA d

Li11 FYSHSAODIOMTOS PSSVSAPIGDRVTITCRASOEIANYLAWYOOKPG KAPK 278 LLIYDTYTLOTDV PPRFSGSGSGTDF SSLOPE DTATYFCOOADI FPLS GGTKWEIKRTWAA d

Li10 FYSHSAODIOMTOS PSSMSASWGDTW TITCRASOGIGNWLAWYOOKPG KAP 279 TLLIYAASSLESGW PSRFTGSGSSSG DF LTIS DL HPEDLATYYCOOAOTF PLT FGGGTRWDLKRTWAAP

iO1 FYSHSAODIOMTOS PSSLSASWGDRW TITCOASO DISNYLNWYOOKPG KA PK 28O LIYDASNLETGWPSRFSGSGSGTDFTL SSLOPE DFATYYCOOADR PAVTE GGGTKWEIKRTWAAP

LiO7 YS SAOSELTOPPSVSVSPGOTAII PWLWI 281 YLDIKRPAGISERFSGSNSGNTATLT CQAMDEA TKLTVLSOPKAAP

LiO3 FYSHSAODIOMTOSPSSLSASVGDRVT CITCRASOSISSYLNWYOOKPG KAP 282 IYAASS OSGVPSRFSGSGSGTDFTL TISSLOPED FATYYCOOSYST PWT GTKWEIKRTWAAP

1Af PAIMSASPGEKW CMTCSASSSVSYMHWYOOKSGTSPKRWIY DTSK 283 LASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCOOWSSNPFTFGSGTKLEI

QMTOS PASLSASWGETW CITCRASGNIYNYLAWFOOKOGKSPOLLWYNAK 284 TLPDGVPSRFSGSGSGTOYFLKINSLOPEDFGSYYCOHFWAIPYTFGGGTKLE IKR

DIVMTOS PSSLTWTAGEKW CMSCKSSOSLLNSGNOKNYLTWYOOKPGOPPK 285 LLIYWASTRESGVPDRFTGSGSGTDFTLTINSWOAEDLAVYYCONDYSYPLFH T FGSGTKLEIR

DIVMTOS PSSLTWTAGEKW CMSCKSSOSLLNSGNOKSYLTWYOOKPGOPPK 286 LLIYWASTRESGVPDRFTGSGSGTDFTLTINSWOAEDLAVYYCONDYSYPL TFGSGTKLEIR

Li13 DIOMTOS PATLSLSPGERA (LSCRASQSWSSYLAWYOOKPGQAPRLLIYDAS 373 NRATGIPARFSGSGSGTDF CLTISSLEPEDFAVYYCOORSNWPMYTFGOGTK EIK US 8,551,476 B2 67 68 TABLE 8- continued VL Polypeptide Sequences

SEQ ID WL Sequence NO :

Li32 DIOMTOSPDSLSASVGDRVTITCOASODISYYLNWYOOKPGMAPKLLIYDA 377 FILEGGAPSRFSGSGSGTDFSFTISNLOPEDIATYFCOOSDOLPWTFGOGTKVE IR

Li33 DIOMTOSPGTLSLSPGERATLSCRASQSWSSYLAWYOOKPGQAPRLLIYDAS 381 NRATGIPARFSGSGSGTEFTLTISSLOSEDFAVYYCOQYDKWPLTFGGGTKV ELK

Li34 DIOMTOSPSSLSASVGDRVTITCHASODISNYLSWYOOKPGKAPKLLIYDAF 385 NLETGVPSRFSGSGSGTDFTFTISSLOPEDFATYYCOHYDNLPFTFGPGTRVA IR

In a further embodiment, the present invention includes an 29-E07 (LiO7), 34-G04 (LiO8), 36-A12 (LiO9), 28-D02 isolated polynucleotide comprising, consisting essentially of (Li10), 30-B01 (Li11), 34-B03 (Li12), Li13, Li32, Li33, or consisting of a nucleic acid encoding a VL at least 80%, Li34, 3383 (L1a. 1), 3495 (L1 a.2), 3563 (L1 a.3), 3564 85%, 90% or 95% identical to a reference VL polypeptide (L1 a.4), 3565 (L1 a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 sequence selected from the group consisting of SEQID NOs: (L1a.8), 3569 (L1a.9), 3570 (L1a. 10), 3571 (L1a. 11), 3582 273 to 286, 373, 377, 381 and 385. In certain embodiments, (L1a. 12), and 1968 (L1 a.13), or will competitively inhibit an antibody or antigen-binding fragment comprising the VL 25 such a monoclonal antibody from binding to Sp35. encoded by the polynucleotide specifically or preferentially In certain embodiments, an antibody or antigen-binding binds to Sp35. fragment thereof comprising, consisting essentially of or In another aspect, the present invention includes an isolated consisting of a VL encoded by one or more of the polynucle polynucleotide comprising, consisting essentially of, or con otides described above specifically or preferentially binds to sisting of a nucleic acid sequence encoding a VL of the 30 an Sp35 polypeptide or fragment thereof, or a Sp35 variant invention, selected from the group consisting of SEQIDNOs: polypeptide, with an affinity characterized by a dissociation 273 to 286, 373, 377,381 and 385. In certain embodiments, constant (Ki) no greater than 5x10°M, 10° M. 5x10 M, an antibody or antigen-binding fragment comprising the VL 10 M, 5x10 M, 10 M, 5x10: M, 10: M, 5x10 M, encoded by the polynucleotide specifically or preferentially 106 M, 5x10-7 M, 10-7 M, 5x10-8M, 108 M, 5x10 M, binds to Sp35. 35 10 M, 5x100M, 100M, 5x10' M, 10 M, 5x10'? In certain embodiments, an antibody or antigen-binding M, 10° M, 5x10' M, 10 M, 5x10 M, 10 M, fragment thereof comprising, consisting essentially of, or 5x10-15 M, or 105 M. consisting of a VL encoded by one or more of the polynucle In additional embodiments, the present invention includes otides described above specifically or preferentially binds to an isolated polynucleotide which encodes a light chain vari the same epitope as a monoclonal antibody selected from the 40 able region (V), where the polynucleotide comprises a V group consisting of 201'. 3A3, 3A6, 1A7, 1G7. 2B10, 2C11. nucleic acid sequence selected from the group consisting of SEQID NOs 185 to 194,371,375,379 and 383, as shown in Table 9. In certain embodiments, an antibody or antigen binding fragment comprising the VL encoded by the poly nucleotide specifically or preferentially binds to Sp35. TABLE 9 VL Polynucleotide Sequences

SEO ID WL Sequence NO :

LiO2 TTCTATTCTCACAGTGCACAGTACGAATTGACTCAGCCACCCTCAGTGTCCGTGTC 185 CCCAGGACAGACAGCCAGCATCACCTGCTCTGGAGATAAATTGGGGGATAAATTTG CTTCCTGGTATCAGCAGAAGGCAGGCCAGTCCCCTGTGCTGGTCATCTTTCAAGAT AGGAAGCGTCTCTCAGGGATCCCTGAGCGATTCTCTCGCTCCAACTCTGGGAACAC AGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGACTATTACTGTC AGGCGTGGGACACCAACACTGTGGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTA GGTCAGCCCAAGGCTGCCCCC

LiO9 TTCTATTCTCACAGTGCACAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTC 186 TGCATTTGTGGGAGACAGAGTCGCCATCACTTGCCGCGCAAGTCAGAGCATCGACA CCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTAT GCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGCAGTGGAACTGG GACAGATTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGGAACTTACT ACTGTCAACAGAGTTACAGTCCCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAG ATCAAACGAACTGTGGCTGCACCA

US 8,551,476 B2 71 72 TABLE 9- continued VL Polynucleotide Sedulences

SEQ ID WL Sequence NO :

Li22 GACATCCAGATGACCCAGTCTCCAGACTCCCTGTC GCATCTGTTGGAGACAGAG 375 TCACCATCACTTGCCAGGCGAGTCAAGACATTAGC ACTATTTAAATTGG ATCA GCAGAAACCAGGGATGGCCCCTAAACTCCTCATCTACGATGCCTTCATTT GGAA GGAGGGGCCCCATCACGGTTCAGTGGGAGCGGCTC GGGACAGATTTTTC TTCA CCATCAGCAATCTACAGCCTGAGGATATTGCAACT ATTTCTGTCAACAG CTGA TCAACTGCCCGTGACCTTCGGCCAAGGGACCAAGGTGOAAATCAGA

Li23 GACATCCAGATGACCCAGTCTCCAGGCACCCTGTC TTGTCTCCAGGGGAAAGAG 379 CCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGG ACCA ACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCC ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTC GGGACAGAGTTCAC CTCA CCATCAGCAGCCTGCAGTCTGAGGATTTTGCAGTT ATTACTGTCAGCAG ATGA TAAGTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

Li24 GACATCCAGATGACCCAGTCTCCATCCTCCCTCTC GCATCTGTAGGAGACAGAG 383 TCACCATCACTTGCCATGCGAGTCAGGACATTAGCAACTATTTAAGTTGG ATCA GCAGAAACCAGGTAAAGCCCCTAAACTCCTGATCTACGATGCTTTCAATT GGAG ACAGGAGTCCCATCGAGGTTCAGTGGAAGTGGATC (OGCACAGATTTTACATTCA CCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACATATTACTGTCAGCAC ATCA TAATCTCCCATTCACTTTCGGCCCTGGGACCAGAGTGGCGATCAGA

In a further embodiment, the present invention includes an 25 Also, as described in more detail elsewhere herein, the isolated polynucleotide comprising, consisting essentially of present invention includes compositions comprising the or consisting of a nucleic acid encoding a VL at least 80%, polynucleotides comprising one or more of the polynucle 85%, 90%, or 95% identical to a VL polynucleotide selected otides described above. In one embodiment, the invention from the group consisting of SEQ ID NOs: 185-194, 371, includes compositions comprising a first polynucleotide and 375, 379 and 383 of Table 9. In certain embodiments, the 30 second polynucleotide wherein said first polynucleotide polynucleotide encodes a VL polypeptide which specifically encodes a VH polypeptide as described herein and wherein or preferentially binds to Sp35. said second polynucleotide encodes a VL polypeptide as In certain embodiments, an antibody or antigen-binding described herein. Specifically a composition which com fragment thereof comprising, consisting essentially of, or 35 prises, consists essentially of, or consists of a VH polynucle consisting of a VL encoded by one or more of the polynucle otide, as show in Table 7, and a VL polynucleotide, as shown otides described above specifically or preferentially binds to in Table 9, wherein said VH polynucleotide and said VL the same epitope as a monoclonal antibody selected from the polynucleotide are selected from the group consisting of group consisting of 201'. 3A3, 3A6, 1A7, 1G7. 2B10, 2C11. i) SEQID NO:173 and SEQID NO:185: 2F3, 3P1D10.2C3, 3P1E1 1.3B7, 3P2C6.3G10.2H7, 40 ii) SEQID NO:174 and SEQID NO:186: 3P2C9.2G4, 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, iii) SEQID NO: 175 and SEQ ID NO: 187: 3P4C5.1 D8, 3P4C8.2G9, 30-C12 (L100), 38-D01 (LiO2), iv) SEQID NO:176 and SEQID NO: 188: 35-E04 (LiO3), 36-C09 (LiO4), 30-A11 (LiO5), 34-F02 v) SEQID NO:178 and SEQID NO:189: (LiO6), 29-E07 (LiO7), 34-G04 (LiO8), 36-A12 (LiO9), vi) SEQID NO: 179 and SEQID NO: 190; 28-D02 (Li10), 30-B01 (Li11), 34-B03 (Li12), Li13, Li32, 45 vii) SEQID NO:180 and SEQID NO:191; Li33, Li34, 3383 (L1a.1), 3495 (L1 a.2), 3563 (L1 a.3), 3564 viii) SEQID NO:181 and SEQID NO: 192: (L1 a.4), 3565 (L1 a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 ix) SEQID NO:182 and SEQID NO:193; (L1a.8), 3569 (L1a.9), 3570 (L1a. 10), 3571 (L1a. 11), 3582 x) SEQID NO:183 and SEQID NO:194; (L1a. 12), and 1968 (L1 a.13), or will competitively inhibit xi) SEQID NO:370 and SEQID NO:371; such a monoclonal antibody from binding to Sp35. 50 xii) SEQID NO:374 and SEQID NO:375; In certain embodiments, an antibody or antigen-binding xiii) SEQID NO:378 and SEQID NO:379; and fragment thereof comprising, consisting essentially of, or xiv) SEQID NO:382 and SEQID NO:385. consisting of a VL encoded by one or more of the polynucle The present invention also includes fragments of the poly otides described above specifically or preferentially binds to nucleotides of the invention, as described elsewhere. Addi an Sp35 polypeptide or fragment thereof, or a Sp35 variant 55 tionally polynucleotides which encode fusion polynucle polypeptide, with an affinity characterized by a dissociation otides, Fab fragments, and other derivatives, as described constant (Ki) no greater than 5x10° M, 10° M, 5x10 M, herein, are also contemplated by the invention. 10 M, 5x10 M, 10 M, 5x10: M, 10: M, 5x10 M, The polynucleotides may be produced or manufactured by 106 M, 5x10-7 M, 10-7 M, 5x108 M, 108 M., 5x10 M, any method known in the art. For example, if the nucleotide 10 M, 5x100M, 100M, 5x10' M, 10 M, 5x10"? 60 sequence of the antibody is known, a polynucleotide encod M, 10-12 M, 5x10-3 M, 103 M., 5x10 M, 10 M, ing the antibody may be assembled from chemically synthe 5x10's M, or 10 M. sized oligonucleotides (e.g., as described in Kutmeier et al., Any of the polynucleotides described above may further BioTechniques 17:242 (1994)), which, briefly, involves the include additional nucleic acids, encoding, e.g., a signal pep synthesis of overlapping oligonucleotides containing por tide to direct secretion of the encoded polypeptide, antibody 65 tions of the sequence encoding the antibody, annealing and constant regions as described herein, or other heterologous ligating of those oligonucleotides, and then amplification of polypeptides as described herein. the ligated oligonucleotides by PCR. US 8,551,476 B2 73 74 Alternatively, a polynucleotide encoding an Sp35 anti diated mutagenesis. Preferably, conservative amino acid Sub body, or antigen-binding fragment, variant, or derivative stitutions are made at one or more non-essential amino acid thereof may be generated from nucleic acid from a suitable residues. Source. If a clone containing a nucleic acid encoding a par ticular antibody is not available, but the sequence of the V. Sp35 Antibody Polypeptides antibody molecule is known, a nucleic acid encoding the antibody may be chemically synthesized or obtained from a The present invention is further directed to isolated suitable source (e.g., an antibody cDNA library, or a cDNA polypeptides which make up Sp35 antibodies, antigen bind library generated from, or nucleic acid, preferably poly ing fragments, variants or derivatives thereof. Sp35 antibod A+RNA, isolated from, any tissue or cells expressing the 10 ies of the present invention comprise polypeptides, e.g., antibody or other Sp35 antibody, such as hybridoma cells amino acid sequences encoding Sp35-specific antigen bind selected to express an antibody) by PCR amplification using ing regions derived from immunoglobulin molecules. A synthetic primers hybridizable to the 3' and 5' ends of the polypeptide or amino acid sequence "derived from a desig sequence or by cloning using an oligonucleotide probe spe nated protein refers to the origin of the polypeptide. In certain cific for the particular gene sequence to identify, e.g., a cDNA 15 cases, the polypeptide or amino acid sequence which is clone from a cDNA library that encodes the antibody or other derived from a particular starting polypeptide or amino acid Sp35 antibody. Amplified nucleic acids generated by PCR sequence has an amino acid sequence that is essentially iden may then be cloned into replicable cloning vectors using any tical to that of the starting sequence, or a portion thereof, method well known in the art. wherein the portion consists of at least 10-20 amino acids, at Once the nucleotide sequence and corresponding amino least 20-30 amino acids, at least 30-50 amino acids, or which acid sequence of the Sp35 antibody, or antigen-binding frag is otherwise identifiable to one of ordinary skill in the art as ment, variant, or derivative thereof is determined, its nucle having its origin in the starting sequence. otide sequence may be manipulated using methods well In one embodiment, the present invention provides an iso known in the art for the manipulation of nucleotide lated polypeptide comprising, consisting essentially of, or sequences, e.g., recombinant DNA techniques, site directed 25 consisting of an immunoglobulin heavy chain variable region mutagenesis, PCR, etc. (see, for example, the techniques (VH), where at least one of CDRs of the heavy chain variable described in Sambrook et al., Molecular Cloning, A Labora region or at least two of the CDRs of the heavy chain variable tory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold region are at least 80%, 85%, 90% or 95% identical to refer Spring Harbor, N.Y. (1990) and Ausubel et al., eds. Current ence heavy chain CDR1, CDR2 or CDR3 amino acid Protocols in Molecular Biology, John Wiley & Sons, NY 30 sequences from monoclonal Sp35 antibodies disclosed (1998), which are both incorporated by reference herein in herein. Alternatively, the CDR1, CDR2 and CDR3 regions of their entireties), to generate antibodies having a different the VH are at least 80%, 85%, 90% or 95% identical to amino acid sequence, for example to create amino acid Sub reference heavy chain CDR1, CDR2 and CDR3 amino acid stitutions, deletions, and/or insertions. sequences from monoclonal Sp35 antibodies disclosed A polynucleotide encoding an Sp35 antibody, or antigen 35 herein. Thus, according to this embodiment a heavy chain binding fragment, variant, or derivative thereof can be com variable region of the invention has CDR1, CDR2, and CDR3 posed of any polyribonucleotide or polydeoxyribonucleotide, polypeptide sequences related to the groups shown in Table 4. which may be unmodified RNA or DNA or modified RNA or Supra. In certain embodiments, an antibody or antigen-bind DNA. For example, a polynucleotide encoding Sp35 anti ing fragment comprising the VH polypeptide specifically or body, or antigen-binding fragment, variant, or derivative 40 preferentially binds to Sp35. thereof can be composed of single- and double-stranded In another embodiment, the present invention provides an DNA, DNA that is a mixture of single- and double-stranded isolated polypeptide comprising, consisting essentially of or regions, single- and double-stranded RNA, and RNA that is consisting of an immunoglobulin heavy chain variable region mixture of single- and double-stranded regions, hybrid mol (VH) in which the CDR1, CDR2, and CDR3 regions have ecules comprising DNA and RNA that may be single 45 polypeptide sequences which are identical to the CDR1. Stranded or, more typically, double-stranded or a mixture of CDR2, and CDR3 groups shown in Table 4. In certain single- and double-stranded regions. In addition, a polynucle embodiments, an antibody or antigen-binding fragment com otide encoding an Sp35 antibody, or antigen-binding frag prising the VH polypeptide specifically or preferentially ment, variant, or derivative thereof can be composed of triple binds to Sp35. stranded regions comprising RNA or DNA or both RNA and 50 In a further embodiment, the present invention includes an DNA. A polynucleotide encoding an Sp35 antibody, or anti isolated polypeptide comprising, consisting essentially of or gen-binding fragment, variant, or derivative thereof may also consisting of a VH polypeptide at least 80%, 85%, 90% 95% contain one or more modified bases or DNA or RNA back or 100% identical to a reference VH polypeptide sequence bones modified for stability or for other reasons. “Modified selected from the group consisting of SEQ ID NOS:158 to bases include, for example, tritylated bases and unusual bases 55 172, 372, 376, 380 and 384 as shown in Table 6. In certain Such as inosine. A variety of modifications can be made to embodiments, an antibody or antigen-binding fragment com DNA and RNA; thus, “polynucleotide' embraces chemically, prising the VH polypeptide specifically or preferentially enzymatically, or metabolically modified forms. binds to Sp35. An isolated polynucleotide encoding a non-natural variant In another aspect, the present invention includes an isolated of a polypeptide derived from an immunoglobulin (e.g., an 60 polypeptide comprising, consisting essentially of, or consist immunoglobulin heavy chain portion or light chain portion) ing of a VH polypeptide selected from the group consisting of can be created by introducing one or more nucleotide Substi SEQID NOs: 158 to 172,372,376, 380 and 384 as shown in tutions, additions or deletions into the nucleotide sequence of Table 6. In certain embodiments, an antibody or antigen the immunoglobulin Such that one or more amino acid Sub binding fragment comprising the VH polypeptide specifically stitutions, additions or deletions are introduced into the 65 or preferentially binds to Sp35. encoded protein. Mutations may be introduced by standard In certain embodiments, an antibody or antigen-binding techniques, such as site-directed mutagenesis and PCR-me fragment thereof comprising, consisting essentially of or US 8,551,476 B2 75 76 consisting of a one or more of the VH polypeptides described binding fragment comprising the VL polypeptide specifically above specifically or preferentially binds to the same epitope or preferentially binds to Sp35. as a monoclonal antibody selected from the group consisting In certain embodiments, an antibody or antigen-binding of 201", 3A3, 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, fragment thereof comprising, consisting essentially of one or 3P1D10.2C3, 3P1E11.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, more of the VL polypeptides described above specifically or 3P4A6.1D9, 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1 D8, preferentially binds to the same epitope as a monoclonal 3P4C8.2G9, 30-C12 (L100), 38-D01 (LiO2), 35-E04 (LiO3), antibody selected from the group consisting of 201'. 3A3, 36-C09 (LiO4), 30-A11 (LiO5), 34-F02 (LiO6), 29-E07 3A6, 1A7, 1G7, 2B10, 2C11, 2F3, 3P1D10.2C3, (LiO7), 34-G04 (Li08), 36-A12 (Li09), 28-D02 (Li10), 3P1E1 1.3B7, 3P2C6.3G10.2H7, 3P2C9.2G4, 3P4A6.1D9, 30-B01 (Li11), 34-B03 (Li12), Li13, Li32, Li33, Li34,3383 10 3P4A1.2B9, 3P4C2.2D2, 3P4C5.1 D8, 3P4C8.2G9, 30-C12 (L1a.1), 3495 (L1 a.2), 3563 (L1 a.3), 3564 (Lla.4), 3565 (L100), 38-D01 (LiO2), 35-E04 (LiO3), 36-C09 (LiO4), (L1 a.5), 3566 (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569 30-A11 (LiO5), 34-F02 (LiO6), 29-E07 (LiO7), 34-G04 (L1a.9), 3570 (L1a. 10), 3571 (L1a. 11), 3582 (L1a.12), and (LiO8), 36-A12 (LiO9), 28-D02 (Li10), 30-B01 (Li11), 1968 (L1a. 13), or will competitively inhibit such a mono 34-B03 (Li12), Li13, Li32, Li33, Li34, 3383 (L1a.1), 3495 clonal antibody from binding to Sp35. 15 (L1 a.2), 3563 (L1 a.3), 3564 (Lla.4), 3565 (L1 a.5), 3566 In certain embodiments, an antibody or antigen-binding (L1a.6), 3567 (L1a.7), 3568 (L1a.8), 3569 (L1a.9), 3570 fragment thereof comprising, consisting essentially of, or (L1a. 10), 3571 (L1a. 11), 3582 (L1a.12), and 1968 (L1a. 13), consisting of one or more of the VH polypeptides described or will competitively inhibit such a monoclonal antibody above specifically or preferentially binds to an Sp35 polypep from binding to Sp35. tide or fragment thereof, or a Sp35 variant polypeptide, with In certain embodiments, an antibody or antigen-binding an affinity characterized by a dissociation constant (Ki) no fragment thereof comprising, consisting essentially of or greater than 5x10°M, 10°M,5x10M, 10 M,5x10M, consisting of a one or more of the VL polypeptides described 10 M, 5x10-S M, 10-s M, 5x106 M, 106 M, 5x10-7 M, above specifically or preferentially binds to an Sp35 polypep 107 M., 5x10 M, 10 M, 5x10M, 10 M, 5x100 M, tide or fragment thereof, or a Sp35 variant polypeptide, with 10-10M,5x10-11M, 10 M,5x10-12M, 10-12M,5x10-13 25 an affinity characterized by a dissociation constant (K) no M, 10-3M, 5x10 M, 10 M, 5x10's M, or 105 M. greater than 5x10M, 10 M,5x10M, 10 M,5x10M, In another embodiment, the present invention provides an 10 M, 5x10: M, 10: M, 5x10 M, 10 M, 5x107 M, isolated polypeptide comprising, consisting essentially of or 107 M., 5x108 M, 10-8M, 5x10 M, 10 M, 5x100 M, consisting of an immunoglobulin light chain variable region 100M,5x10-11M, 10 M,5x10-12M, 10-12M,5x10-13 (VL), where at least one of the CDRs of the light chain 30 M, 10-3M, 5x10 M, 10 M, 5x10's M, or 10-15 M. variable region or at least two of the CDRs of the light chain In other embodiments, an antibody or antigen-binding variable region are at least 80%, 85%, 90% or 95% identical fragment thereof comprises, consists essentially of or con to reference heavy chain CDR1, CDR2, or CDR3 amino acid sists of a VH polypeptide, as shown in Table 6, and a VL sequences from monoclonal Sp35 antibodies disclosed polypeptide, as shown in Table 8, selected from the group herein. Alternatively, the CDR1, CDR2 and CDR3 regions of 35 consisting of: the VL are at least 80%, 85%, 90% or 95% identical to i) SEQID NO: 170 and SEQID NO:283: reference light chain CDR1, CDR2, and CDR3 amino acid ii) SEQID NO:171 and SEQID NO:284; sequences from monoclonal Sp35 antibodies disclosed iii) SEQID NO: 172 and SEQ ID NO:285: herein. Thus, according to this embodiment a light chain iv) SEQID NO: 172 and SEQID NO:286; variable region of the invention has CDR1, CDR2, and CDR3 40 v) SEQID NO:158 and SEQID NO:273; polypeptide sequences related to the polypeptides shown in vi) SEQID NO: 159 and SEQID NO:274; Table 5. Supra. In certain embodiments, an antibody or anti vii) SEQID NO:160 and SEQID NO:275: gen-binding fragment comprising the VL polypeptide spe viii) SEQID NO:161 and SEQID NO:276; cifically or preferentially binds to Sp35. ix) SEQID NO: 163 and SEQID NO:277; In another embodiment, the present invention provides an 45 x) SEQID NO:164 and SEQID NO:278; isolated polypeptide comprising, consisting essentially of or xi) SEQID NO: 165 and SEQID NO:279; consisting of an immunoglobulin light chain variable region xii) SEQID NO:166 and SEQID NO:280; (VL) in which the CDR1, CDR2, and CDR3 regions have xiii) SEQID NO:167 and SEQID NO:281; polypeptide sequences which are identical to the CDR1. xiv) SEQID NO:168 and SEQID NO:282; CDR2, and CDR3 groups shown in Table 5. In certain 50 xv) SEQ ID NO:372 and SEQID NO:373; embodiments, an antibody or antigen-binding fragment com xvi) SEQID NO:376 and SEQID NO:377; prising the VL polypeptidespecifically or preferentially binds xvii) SEQID NO:380 and SEQID NO:381; and to Sp35. xviii) SEQID NO:384 and SEQID NO:385. In a further embodiment, the present invention includes an Any of the polypeptides described above may further isolated polypeptide comprising, consisting essentially of or 55 include additional polypeptides, e.g., a signal peptide to consisting of a VL polypeptide at least 80%, 85%, 90% or direct secretion of the encoded polypeptide, antibody con 95% identical to a reference VL polypeptide sequence stant regions as described herein, or other heterologous selected from the group consisting of SEQ ID NOS:273 to polypeptides as described herein. Additionally, polypeptides 286, 373, 377, 381 and 385, shown in Table 8. In certain of the invention include polypeptide fragments as described embodiments, an antibody or antigen-binding fragment com 60 elsewhere. Additionally polypeptides of the invention include prising the VL polypeptidespecifically or preferentially binds fusion polypeptide, Fab fragments, and other derivatives, as to Sp35. described herein. In another aspect, the present invention includes an isolated Also, as described in more detail elsewhere herein, the polypeptide comprising, consisting essentially of, or consist present invention includes compositions comprising the ing of a VL polypeptide selected from the group consisting of 65 polypeptides described above. SEQID NOS: 273 to 286, 373, 377, 381 and 385, shown in It will also be understood by one of ordinary skill in the art Table 8. In certain embodiments, an antibody or antigen that Sp35 antibody polypeptides as disclosed herein may be US 8,551,476 B2 77 78 modified Such that they vary in amino acid sequence from the having a similar side chain. Families of amino acid residues naturally occurring binding polypeptide from which they having similar side chains have been defined in the art, includ were derived. For example, a polypeptide or amino acid ing basic side chains (e.g., lysine, arginine, histidine), acidic sequence derived from a designated protein may be similar, side chains (e.g., aspartic acid, glutamic acid), uncharged e.g., have a certain percent identity to the starting sequence, polar side chains (e.g., glycine, asparagine, glutamine, serine, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, or 95% threonine, tyrosine, cysteine), nonpolar side chains (e.g., ala identical to the starting sequence. nine, Valine, leucine, isoleucine, proline, phenylalanine, Furthermore, nucleotide oramino acid substitutions, dele methionine, tryptophan), beta-branched side chains (e.g., tions, or insertions leading to conservative Substitutions or threonine, Valine, isoleucine) and aromatic side chains (e.g., changes at “non-essential amino acid regions may be made. 10 tyrosine, phenylalanine, tryptophan, histidine). Thus, a non For example, a polypeptide or amino acid sequence derived essential amino acid residue in an immunoglobulin polypep from a designated protein may be identical to the starting tide is preferably replaced with another amino acid residue sequence except for one or more individual amino acid Sub from the same side chain family. In another embodiment, a stitutions, insertions, or deletions, e.g., one, two, three, four, string of amino acids can be replaced with a structurally five, six, seven, eight, nine, ten, fifteen, twenty or more indi 15 similar string that differs in order and/or composition of side vidual amino acid Substitutions, insertions, or deletions. In chain family members. certain embodiments, a polypeptide or amino acid sequence Alternatively, in another embodiment, mutations may be derived from a designated protein has one to five, one to ten, introduced randomly along all or part of the immunoglobulin one to fifteen, or one to twenty individual amino acid substi coding sequence, such as by Saturation mutagenesis, and the tutions, insertions, or deletions relative to the starting resultant mutants can be incorporated into Sp35 antibodies Sequence. for use in the diagnostic and treatment methods disclosed Certain Sp35 antibody polypeptides of the present inven herein and screened for their ability to bind to the desired tion comprise, consist essentially of, or consist of an amino antigen, e.g., Sp35. acid sequence derived from a human amino acid sequence. However, certain Sp35 antibody polypeptides comprise one 25 VI. Fusion Proteins and Antibody Conjugates or more contiguous amino acids derived from another mam malian species. For example, an Sp35 antibody of the present As discussed in more detail elsewhere herein, Sp35 anti invention may include a primate heavy chain portion, hinge bodies, or antigen-binding fragments, variants, or derivatives portion, orantigenbinding region. In another example, one or thereof of the invention may further be recombinantly fused more murine-derived amino acids may be present in a non 30 to a heterologous polypeptide at the N- or C-terminus or murine antibody polypeptide, e.g., in an antigen binding site chemically conjugated (including covalent and non-covalent of an Sp35 antibody. In certain therapeutic applications, conjugations) to polypeptides or other compositions. For Sp35-specific antibodies, or antigen-binding fragments, vari example, Sp35-specific Sp35 antibodies may be recombi ants, or analogs thereof are designed so as to not be immu nantly fused or conjugated to molecules useful as labels in nogenic in the animal to which the antibody is administered. 35 detection assays and effector molecules Such as heterologous In certain embodiments, an Sp35 antibody polypeptide polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT comprises an amino acid sequence or one or more moieties publications WO92/08495; WO 91/14438: WO 89/12624; not normally associated with an antibody. Exemplary modi U.S. Pat. No. 5,314.995; and EP396.387, which are incorpo fications are described in more detail below. For example, a rated herein by reference in their entireties. single-chain fiv antibody fragment of the invention may com 40 Sp35 antibodies, orantigen-binding fragments, variants, or prise a flexible linker sequence, or may be modified to add a derivatives thereof of the invention include derivatives that functional moiety (e.g., PEG, a drug, a toxin, or a label). are modified, i.e., by the covalent attachment of any type of An Sp35 antibody polypeptide of the invention may com molecule to the antibody such that covalent attachment does prise, consist essentially of, or consist of a fusion protein. not prevent the antibody binding Sp35. For example, but not Fusion proteins are chimeric molecules which comprise, for 45 by way of limitation, the antibody derivatives include anti example, an immunoglobulin antigen-binding domain with at bodies that have been modified, e.g., by glycosylation, acety least one target binding site, and at least one heterologous lation, pegylation, phosphylation, phosphorylation, amida portion, i.e., a portion with which it is not naturally linked in tion, derivatization by known protecting/blocking groups, nature. The amino acid sequences may normally exist in proteolytic cleavage, linkage to a cellular ligand or other separate proteins that are brought together in the fusion 50 protein, etc. Any of numerous chemical modifications may be polypeptide or they may normally exist in the same protein carried out by known techniques, including, but not limited to but are placed in a new arrangement in the fusion polypeptide. specific chemical cleavage, acetylation, formylation, meta Fusion proteins may be created, for example, by chemical bolic synthesis of tunicamycin, etc. Additionally, the deriva synthesis, or by creating and translating a polynucleotide in tive may contain one or more non-classical amino acids. which the peptide regions are encoded in the desired relation 55 Sp35 antibodies, orantigen-binding fragments, variants, or ship. derivatives thereof of the invention can be composed of amino The term "heterologous as applied to a polynucleotide or acids joined to each other by peptide bonds or modified pep a polypeptide, means that the polynucleotide or polypeptide tide bonds, i.e., peptide isosteres, and may contain amino is derived from a distinct entity from that of the rest of the acids other than the 20 gene-encoded amino acids. Sp35 entity to which it is being compared. For instance, as used 60 specific antibodies may be modified by natural processes, herein, a "heterologous polypeptide' to be fused to an Sp35 Such as posttranslational processing, or by chemical modifi antibody, or an antigen-binding fragment, variant, or analog cation techniques which are well known in the art. Such thereof is derived from a non-immunoglobulin polypeptide of modifications are well described in basic texts and in more the same species, or an immunoglobulin or non-immunoglo detailed monographs, as well as in a Voluminous research bulin polypeptide of a different species. 65 literature. Modifications can occur anywhere in the Sp35 A “conservative amino acid substitution' is one in which specific antibody, including the peptide backbone, the amino the amino acid residue is replaced with an amino acid residue acid side-chains and the amino or carboxyl termini, or on US 8,551,476 B2 79 80 moieties such as carbohydrates. It will be appreciated that the of an Sp35-specific antibody and the amino acid sequence of same type of modification may be present in the same or any one, two, three or more of the V, CDRs of an Sp35 varying degrees at several sites in a given Sp35-specific anti specific antibody, or fragments or variants thereof, and a body. Also, a given Sp35-specific antibody may contain many heterologous polypeptide sequence. Preferably, two, three, types of modifications. Sp35-specific antibodies may be four, five, six, or more of the VCDR(s) or V, CDR(s) corre branched, for example, as a result of ubiquitination, and they spond to single source antibody (or ScFv or Fab fragment) of may be cyclic, with or without branching. Cyclic, branched, the invention. Nucleic acid molecules encoding these fusion and branched cyclic Sp35-specific antibodies may result from proteins are also encompassed by the invention. posttranslation natural processes or may be made by synthetic Exemplary fusion proteins reported in the literature methods. Modifications include acetylation, acylation, ADP 10 include fusions of the T cell receptor (Gascoigne et al., Proc. ribosylation, amidation, covalent attachment of flavin, cova Natl. Acad. Sci. USA 84:2936-2940 (1987)); CD4 (Capon et lent attachment of a heme moiety, covalent attachment of a al., Nature 337:525-531 (1989); Traunecker et al., Nature nucleotide or nucleotide derivative, covalent attachment of a 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA lipid or lipid derivative, covalent attachment of phosphoti 9:347-353 (1990); and Byrnet al., Nature 344:667-670 dylinositol, cross-linking, cyclization, disulfide bond forma 15 (1990)); L-selectin (homing receptor) (Watson et al., J. Cell. tion, demethylation, formation of covalent cross-links, for Biol. 110:2221-2229 (1990); and Watson et al., Nature 349: mation of cysteine, formation of pyroglutamate, formylation, 164-167 (1991)): CD44 (Aruffo et al., Cell 61: 1303-1313 gamma-carboxylation, glycosylation, GPI anchor formation, (1990)); CD28 and B7 (Linsley et al., J. Exp. Med. 173:721 hydroxylation, iodination, methylation, myristoylation, oxi 730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med. 174:561-569 dation, pegylation, proteolytic processing, phosphorylation, (1991)). CD22 (Stamenkovic et al., Cell 66:1133-1144 prenylation, racemization, selenoylation, Sulfation, transfer (1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. RNA mediated addition of amino acids to proteins such as Sci. USA 88:10535-10539 (1991); Lesslauer et al., Eur: J. arginylation, and ubiquitination. (See, for instance, Pro Immunol. 27:2883-2886 (1991); and Peppel et al., J. Exp. teins—Structure And Molecular Properties, T. E. Creighton, Med. 174: 1483-1489 (1991)); and IgE receptor a (Ridgway W. H. Freeman and Company, New York 2nd Ed., (1993); 25 and Gorman, J. Cell. Biol. Vol. 115, Abstract No. 1448 Posttranslational Covalent Modification Of Proteins, B. C. (1991)). Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); In certain embodiments, Sp35 antibodies, antibody frag Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et ments, derivatives and variants thereof further comprise a al., Ann NYAcadSci 663:48-62 (1992)). targeting moiety. Targeting moieties include a protein or a The present invention also provides for fusion proteins 30 peptide which directs localization to a certain part of the body, comprising an Sp35 antibody, or antigen-binding fragment, for example, to the brain or compartments therein. In certain variant, or derivative thereof, and a heterologous polypeptide. embodiments, Sp35 antibodies, antibody fragments, deriva The heterologous polypeptide to which the antibody is fused tives and variants thereof are attached or fused to a brain may be useful for function or is useful to target the Sp35 targeting moiety. The brain targeting moieties are attached polypeptide expressing cells. In one embodiment, a fusion 35 covalently (e.g., direct, translational fusion, or by chemical protein of the invention comprises, consists essentially of or linkage either directly or through a spacer molecule, which consists of a polypeptide having the amino acid sequence of can be optionally cleavable) or non-covalently attached (e.g., any one or more of the V regions of an antibody of the through reversible interactions such as avidin, biotin, protein invention or the amino acid sequence of any one or more of A, IgG, etc.). In other embodiments, the Sp35 antibodies, the V, regions of an antibody of the invention or fragments or 40 antibody fragments, derivatives and variants thereof are variants thereof, and a heterologous polypeptide sequence. In attached to one more brain targeting moieties. In additional another embodiment, a fusion protein for use in the diagnostic embodiments, the brain targeting moiety is attached to a and treatment methods disclosed herein comprises, consists plurality of Sp35 antibodies, antibody fragments, derivatives essentially of, or consists of a polypeptide having the amino and variants thereof. acid sequence of any one, two, three of the V. CDRS of an 45 Abrain targeting moiety associated with an Sp35 antibody, Sp35-specific antibody, or fragments, variants, or derivatives antibody fragment, derivative or variant thereof enhances thereof, or the amino acid sequence of any one, two, three of brain delivery of such an Sp35 antibodies, antibody frag the V, CDRs of an Sp35-specific antibody, or fragments, ments, derivatives and variants thereof. A number of polypep variants, or derivatives thereof, and a heterologous polypep tides have been described which, when fused to a protein or tide sequence. In one embodiment, the fusion protein com 50 therapeutic agent, delivers the protein or therapeutic agent prises a polypeptide having the amino acid sequence of a V through the blood brain barrier (BBB). Non-limiting CDR3 of an Sp35-specific antibody of the present invention, examples include the single domain antibody FC5 (Abulrob or fragment, derivative, or variant thereof, and a heterologous et al. (2005).J. Neurochem. 95, 1201-1214); mAB 83-14, a polypeptide sequence, which fusion protein specifically monoclonal antibody to the human insulin receptor (Par binds to at least one epitope of Sp35. In another embodiment, 55 dridge etal. (1995) Pharmacol. Res. 12,807-816); the B2, B6 a fusion protein comprises a polypeptide having the amino and B8 peptides binding to the human transferrin receptor acid sequence of at least one V region of an Sp35-specific (hTfR) (Xia et al. (2000) J. Virol. 74, 11359-11366); the antibody of the invention and the amino acid sequence of at OX26 monoclonal antibody to the transferrin receptor (Par least oneV, region of an Sp35-specific antibody of the inven dridge etal. (1991).J. Pharmacol. Exp. Ther: 259, 66-70); and tion or fragments, derivatives or variants thereof, and a het 60 SEQ ID NOs: 1-18 of U.S. Pat. No. 6,306,365. The contents erologous polypeptide sequence. Preferably, the V and V, of the above references are incorporated herein by reference regions of the fusion protein correspond to a single source in their entirety. antibody (or schvor Fab fragment) which specifically binds Enhanced brain delivery of an Sp35 antibody, antibody at least one epitope of Sp35. In yet another embodiment, a fragment, derivative or variant thereof is determined by a fusion protein for use in the diagnostic and treatment methods 65 number of means well established in the art. For example, disclosed herein comprises a polypeptide having the amino administering to an animal a radioactively, enzymatically or acid sequence of any one, two, three or more of the VCDRS fluorescently labeled Sp35 antibody, antibody fragment, US 8,551,476 B2 81 82 derivative and variant thereoflinked to a brain targeting moi thereof of the invention conjugated to a diagnostic or thera ety; determining brain localization; and comparing localiza peutic agent. The Sp35 antibodies can be used diagnostically tion with an equivalent radioactively, enzymatically or fluo to, for example, monitor the development or progression of a rescently labeled Sp35 antibody, antibody fragment, neurological disease as part of a clinical testing procedure to, derivative or variant thereofthat is not associated with a brain e.g., determine the efficacy of a given treatment and/or pre targeting moiety. Other means of determining enhanced tar vention regimen. Detection can be facilitated by coupling the geting are described in the above references. Sp35 antibody, or antigen-binding fragment, variant, or As discussed elsewhere herein, Sp35 antibodies, or anti derivative thereof to a detectable substance. Examples of gen-binding fragments, variants, or derivatives thereof of the detectable Substances include various enzymes, prosthetic invention may be fused to heterologous polypeptides to 10 groups, fluorescent materials, luminescent materials, biolu increase the in vivo half life of the polypeptides or for use in minescent materials, radioactive materials, positron emitting immunoassays using methods known in the art. For example, metals using various positron emission tomographies, and in one embodiment, PEG can be conjugated to the Sp35 nonradioactive paramagnetic metal ions. See, for example, antibodies of the invention to increase their half-life in vivo. U.S. Pat. No. 4,741,900 for metal ions which can be conju Leong, S. R., et al., Cytokine 16:106 (2001); Adv. in Drug 15 gated to antibodies for use as diagnostics according to the Deliv: Rev. 54:531 (2002); or Weir et al., Biochem. Soc. Trans present invention. Examples of Suitable enzymes include actions 30:512 (2002). horseradish peroxidase, alkaline phosphatase, B-galactosi Moreover, Sp35 antibodies, or antigen-binding fragments, dase, or acetylcholinesterase; examples of suitable prosthetic variants, orderivatives thereof of the invention can befused to group complexes include Streptavidin/biotin and avidin/bi marker sequences, such as a peptide to facilitate their purifi otin; examples of Suitable fluorescent materials include cation or detection. In preferred embodiments, the marker umbelliferone, fluorescein, fluorescein isothiocyanate, amino acid sequence is a hexa-histidine peptide. Such as the rhodamine, dichlorotriazinylamine fluorescein, dansyl chlo tag provided in a pCRE vector (QIAGEN, Inc., 9259 Eton ride or phycoerythrin; an example of a luminescent material Avenue, Chatsworth, Calif., 91311), among others, many of includes luminol; examples of bioluminescent materials which are commercially available. As described in Gentz et 25 include luciferase, luciferin, and aequorin; and examples of al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for suitable radioactive material include I, I, '''In or 'Tc. instance, hexa-histidine provides for convenient purification An Sp35 antibody, orantigen-binding fragment, variant, or of the fusion protein. Other peptide tags useful for purifica derivative thereof also can be detectably labeled by coupling tion include, but are not limited to, the “HA' tag, which it to a chemiluminescent compound. The presence of the corresponds to an epitope derived from the influenza hemag 30 chemiluminescent-tagged Sp35 antibody is then determined glutinin protein (Wilson et al., Cell 37:767 (1984)) and the by detecting the presence of luminescence that arises during “flag” tag. the course of a chemical reaction. Examples of particularly Fusion proteins can be prepared using methods that are useful chemiluminescent labeling compounds are luminol, well known in the art (see for example U.S. Pat. Nos. 5,116, isoluminol, theromatic acridinium ester, imidazole, acri 964 and 5.225,538). The precise site at which the fusion is 35 dinium salt and oxalate ester. made may be selected empirically to optimize the secretion or One of the ways in which an Sp35 antibody, or antigen binding characteristics of the fusion protein. DNA encoding binding fragment, variant, or derivative thereof can be detect the fusion protein is then transfected into a host cell for ably labeled is by linking the same to an enzyme and using the expression. linked product in an enzyme immunoassay (EIA) (Voller, A., Sp35 antibodies or antigen-binding fragments, variants, or 40 “The Enzyme Linked Immunosorbent Assay (ELISA)' derivatives thereof of the present invention may be used in Microbiological Associates Quarterly Publication, Walkers non-conjugated form or may be conjugated to at least one of ville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. a variety of molecules, e.g., to improve the therapeutic prop Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzy erties of the molecule, to facilitate target detection, or for mol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immu imaging ortherapy of the patient. Sp35 antibodies, orantigen 45 noassay, CRC Press, Boca Raton, Fla., (1980); Ishikawa, E. binding fragments, variants, or derivatives thereof of the et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo invention can be labeled or conjugated either before or after (1981). The enzyme, which is bound to the Sp35 antibody purification, when purification is performed. will react with an appropriate substrate, preferably a chro In particular, Sp35 antibodies, or antigen-binding frag mogenic Substrate, in Such a manner as to produce a chemical ments, variants, or derivatives thereof of the invention may be 50 moiety which can be detected, for example, by spectropho conjugated to therapeutic agents, prodrugs, peptides, pro tometric, fluorimetric or by visual means. Enzymes which teins, enzymes, viruses, lipids, biological response modifiers, can be used to detectably label the antibody include, but are pharmaceutical agents, or PEG. not limited to, malate dehydrogenase, staphylococcal Those skilled in the art will appreciate that conjugates may nuclease, delta-5-steroid isomerase, yeast alcohol dehydro also be assembled using a variety of techniques depending on 55 genase, alpha-glycerophosphate, dehydrogenase, triose the selected agent to be conjugated. For example, conjugates phosphate isomerase, horseradish peroxidase, alkaline phos with biotin are prepared e.g. by reacting a binding polypep phatase, asparaginase, glucose oxidase, beta-galactosidase, tide with an activated ester of biotin such as the biotin N-hy ribonuclease, urease, catalase, glucose-6-phosphate dehydro droxysuccinimide ester. Similarly, conjugates with a fluores genase, glucoamylase and acetylcholinesterase. Additionally, cent marker may be prepared in the presence of a coupling 60 the detection can be accomplished by colorimetric methods agent, e.g. those listed herein, or by reaction with an isothio which employ a chromogenic Substrate for the enzyme. cyanate, preferably fluorescein-isothiocyanate. Conjugates Detection may also be accomplished by visual comparison of of the Sp35 antibodies, or antigen-binding fragments, vari the extent of enzymatic reaction of a Substrate in comparison ants, or derivatives thereof of the invention are prepared in an with similarly prepared Standards. analogous manner. 65 Detection may also be accomplished using any of a variety The present invention further encompasses Sp35 antibod of other immunoassays. For example, by radioactively label ies, or antigen-binding fragments, variants, or derivatives ing the Sp35 antibody, or antigen-binding fragment, variant, US 8,551,476 B2 83 84 or derivative thereof, it is possible to detect the antibody Recombinant expression of an antibody, or fragment, through the use of a radioimmunoassay (RIA) (see, for derivative or analog thereof, e.g., a heavy or light chain of an example, Weintraub, B., Principles of Radioimmunoassays, antibody which binds to a target molecule described herein, Seventh Training Course on Radioligand Assay Techniques, e.g., Sp35, requires construction of an expression vector con The Endocrine Society, (March, 1986)), which is incorpo taining a polynucleotide that encodes the antibody. Once a rated by reference herein). The radioactive isotope can be polynucleotide encoding an antibody molecule or a heavy or detected by means including, but not limited to, a gamma light chain of an antibody, or portion thereof (preferably counter, a Scintillation counter, or autoradiography. containing the heavy or light chain variable domain), of the An Sp35 antibody, orantigen-binding fragment, variant, or invention has been obtained, the vector for the production of derivative thereof can also be detectably labeled using fluo 10 the antibody molecule may be produced by recombinant rescence emitting metals such as 152Eu, or others of the DNA technology using techniques well known in the art. lanthanide series. These metals can be attached to the anti Thus, methods for preparing a protein by expressing a poly body using Such metal chelating groups as diethylenetri nucleotide containing an antibody encoding nucleotide aminepentacetic acid (DTPA) or ethylenediaminetetraacetic sequence are described herein. Methods which are well acid (EDTA). 15 known to those skilled in the art can be used to construct Techniques for conjugating various moieties to an Sp35 expression vectors containing antibody coding sequences and antibody, or antigen-binding fragment, variant, or derivative appropriate transcriptional and translational control signals. thereof are well known, see, e.g., Arnon et al., “Monoclonal These methods include, for example, in vitro recombinant Antibodies For Immunotargeting Of Drugs. In Cancer DNA techniques, synthetic techniques, and in vivo genetic Therapy”, in Monoclonal Antibodies And Cancer Therapy, recombination. The invention, thus, provides replicable vec Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); tors comprising a nucleotide sequence encoding an antibody Hellstrom et al., “Antibodies For Drug Delivery', in Con molecule of the invention, or a heavy or light chain thereof, or trolled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Mar a heavy or light chain variable domain, operably linked to a cel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Car promoter. Such vectors may include the nucleotide sequence riers Of Cytotoxic Agents. In Cancer Therapy: A Review', in 25 encoding the constant region of the antibody molecule (see, Monoclonal Antibodies '84. Biological And Clinical Appli e.g., PCT Publication WO 86/05807; PCT Publication WO cations, Pinchera et al. (eds.), pp. 475-506 (1985): 'Analysis, 89/01036; and U.S. Pat. No. 5,122,464) and the variable Results, And Future Prospective Of The Therapeutic Use Of domain of the antibody may be cloned into such a vector for Radiolabeled Antibody In Cancer Therapy”, in Monoclonal expression of the entire heavy or light chain. Antibodies For Cancer Detection And Therapy, Baldwin et al. 30 The host cell may be co-transfected with two expression (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., vectors of the invention, the first vector encoding a heavy “The Preparation And Cytotoxic Properties Of Antibody chain derived polypeptide and the second vector encoding a Toxin Conjugates. Immunol. Rev. 62:119-58 (1982). light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of VII. Expression of Antibody Polypeptides 35 heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain As is well known, RNA may be isolated from the original polypeptides. In Such situations, the light chain is advanta hybridoma cells or from other transformed cells by standard geously placed before the heavy chain to avoid an excess of techniques, such as guanidinium isothiocyanate extraction toxic free heavy chain (Proudfoot, Nature 322:52 (1986); and precipitation followed by centrifugation or chromatogra 40 Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The phy. Where desirable, mRNA may be isolated from total RNA coding sequences for the heavy and light chains may com by Standard techniques such as chromatography on oligo dT prise cDNA or genomic DNA. cellulose. Suitable techniques are familiar in the art. The term “vector or “expression vector is used herein to In one embodiment, cDNAs that encode the light and the mean vectors used in accordance with the present invention as heavy chains of the antibody may be made, either simulta 45 a vehicle for introducing into and expressing a desired gene in neously or separately, using reverse transcriptase and DNA a host cell. As known to those skilled in the art, such vectors polymerase in accordance with well known methods. PCR may easily be selected from the group consisting of plasmids, may be initiated by consensus constant region primers or by phages, viruses and retroviruses. In general, vectors compat more specific primers based on the published heavy and light ible with the instant invention will comprise a selection chain DNA and amino acid sequences. As discussed above, 50 marker, appropriate restriction sites to facilitate cloning of the PCR also may be used to isolate DNA clones encoding the desired gene and the ability to enter and/or replicate in antibody light and heavy chains. In this case the libraries may eukaryotic or prokaryotic cells. be screened by consensus primers or larger homologous For the purposes of this invention, numerous expression probes, such as mouse constant region probes. vector systems may be employed. For example, one class of DNA, typically plasmid DNA, may be isolated from the 55 vector utilizes DNA elements which are derived from animal cells using techniques known in the art, restriction mapped viruses such as bovine papilloma virus, polyomavirus, aden and sequenced in accordance with standard, well known tech ovirus, vaccinia virus, baculovirus, retroviruses (RSV. niques set forth in detail, e.g., in the foregoing references MMTV or MOMLV) or SV40 virus. Others involve the use of relating to recombinant DNA techniques. Ofcourse, the DNA polycistronic systems with internal ribosome binding sites. may be synthetic according to the present invention at any 60 Additionally, cells which have integrated the DNA into their point during the isolation process or Subsequent analysis. chromosomes may be selected by introducing one or more Following manipulation of the isolated genetic material to markers which allow selection of transfected host cells. The provide Sp35 antibodies, or antigen-binding fragments, Vari marker may provide for prototrophy to an auxotrophic host, ants, or derivatives thereof of the invention, the polynucle biocide resistance (e.g., antibiotics) or resistance to heavy otides encoding the Sp35 antibodies are typically inserted in 65 metals such as copper. The selectable marker gene can either an expression vector for introduction into host cells that may be directly linked to the DNA sequences to be expressed, or be used to produce the desired quantity of Sp35 antibody. introduced into the same cell by cotransformation. Additional US 8,551,476 B2 85 86 elements may also be needed for optimal synthesis of mRNA. electroporation. The host cells harboring the expression con These elements may include signal sequences, splice signals, struct are grown under conditions appropriate to the produc as well as transcriptional promoters, enhancers, and termina tion of the light chains and heavy chains, and assayed for tion signals. heavy and/or light chain protein synthesis. Exemplary assay In particularly preferred embodiments the cloned variable techniques include enzyme-linked immunosorbent assay region genes are inserted into an expression vector along with (ELISA), radioimmunoassay (RIA), or fluorescence-acti the heavy and light chain constant region genes (preferably vated cell sorter analysis (FACS), immunohistochemistry and human) synthetic as discussed above. In one embodiment, the like. this is effected using a proprietary expression vector of Bio The expression vector is transferred to a host cell by con gen IDEC, Inc., referred to as NEOSPLA (U.S. Pat. No. 10 ventional techniques and the transfected cells are then cul 6,159,730). This vector contains the cytomegalovirus pro tured by conventional techniques to produce an antibody for moter/enhancer, the mouse beta globin major promoter, the use in the methods described herein. Thus, the invention SV40 origin of replication, the bovine growth hormone poly includes host cells containing a polynucleotide encoding an adenylation sequence, neomycin phosphotransferase exon 1 antibody of the invention, or a heavy or light chain thereof, and exon 2, the dihydrofolate reductase gene and leader 15 operably linked to a heterologous promoter. In preferred sequence. This vector has been found to result in very high embodiments for the expression of double-chained antibod level expression of antibodies upon incorporation of variable ies, vectors encoding both the heavy and light chains may be and constant region genes, transfection in CHO cells, fol co-expressed in the host cell for expression of the entire lowed by selection in G418 containing medium and methotr immunoglobulin molecule, as detailed below. exate amplification. Of course, any expression vector which As used herein, "host cells' refers to cells which harbor is capable of eliciting expression in eukaryotic cells may be vectors constructed using recombinant DNA techniques and used in the present invention. Examples of suitable vectors encoding at least one heterologous gene. In descriptions of include, but are not limited to plasmids pcDNA3, pHCMV/ processes for isolation of antibodies from recombinant hosts, Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/ the terms “cell and “cell culture' are used interchangeably to Zeo2, pTRACER-HCMV. pUB6/V5-His, pVAX1, and 25 denote the source of antibody unless it is clearly specified pZeoSV2 (available from Invitrogen, San Diego, Calif.), and otherwise. In other words, recovery of polypeptide from the plasmid pCI (available from Promega, Madison, Wis.). In “cells' may mean either from spun down whole cells, or from general, screening large numbers of transformed cells for the cell culture containing both the medium and the Sus those which express suitably high levels if immunoglobulin pended cells. heavy and light chains is routine experimentation which can 30 A variety of host-expression vector systems may be uti be carried out, for example, by robotic systems. Vector sys lized to express antibody molecules for use in the methods tems are also taught in U.S. Pat. Nos. 5,736,137 and 5,658, described herein. Such host-expression systems represent 570, each of which is incorporated by reference in its entirety vehicles by which the coding sequences of interest may be herein. This system provides for high expression levels, e.g., produced and Subsequently purified, but also represent cells >30 pg/cell/day. Other exemplary vector systems are dis 35 which may, when transformed or transfected with the appro closed e.g., in U.S. Pat. No. 6,413,777. priate nucleotide coding sequences, express an antibody mol In other preferred embodiments the Sp35 antibodies, or ecule of the invention in situ. These include but are not limited antigen-binding fragments, variants, or derivatives thereof of to microorganisms such as bacteria (e.g., E. coli, B. subtilis) the invention may be expressed using polycistronic con transformed with recombinant bacteriophage DNA, plasmid structs such as those disclosed in United States Patent Appli 40 DNA or cosmid DNA expression vectors containing antibody cation Publication No. 2003-0157641 A1, filed Nov. 18, 2002 coding sequences; yeast (e.g., Saccharomyces, Pichia) trans and incorporated herein in its entirety. In these novel expres formed with recombinant yeast expression vectors containing sion systems, multiple gene products of interest Such as heavy antibody coding sequences; insect cell systems infected with and light chains of antibodies may be produced from a single recombinant virus expression vectors (e.g., baculovirus) con polycistronic construct. These systems advantageously use 45 taining antibody coding sequences; plant cell systems an internal ribosome entry site (IRES) to provide relatively infected with recombinant virus expression vectors (e.g., cau high levels of Sp35 antibodies, e.g., binding polypeptides, liflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or e.g., Sp35-specific antibodies or immunospecific fragments transformed with recombinant plasmid expression vectors thereof in eukaryotic host cells. Compatible IRES sequences (e.g., Tiplasmid) containing antibody coding sequences; or are disclosed in U.S. Pat. No. 6,193.980 which is also incor 50 mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 porated herein. Those skilled in the art will appreciate that cells) harboring recombinant expression constructs contain Such expression systems may be used to effectively produce ing promoters derived from the genome of mammalian cells the full range of Sp35 antibodies disclosed in the instant (e.g., metallothionein promoter) or from mammalian viruses application. (e.g., the adenovirus late promoter, the vaccinia virus 7.5K More generally, once the vector or DNA sequence encod 55 promoter). Preferably, bacterial cells such as Escherichia ing a monomeric subunit of the Sp35 antibody has been coli, and more preferably, eukaryotic cells, especially for the prepared, the expression vector may be introduced into an expression of whole recombinant antibody molecule, are appropriate host cell. Introduction of the plasmid into the host used for the expression of a recombinant antibody molecule. cell can be accomplished by various techniques well known For example, mammalian cells Such as Chinese hamster to those of skill in theart. These include, but are not limited to, 60 ovary cells (CHO), in conjunction with a vector such as the transfection (including electrophoresis and electroporation), major intermediate early gene promoter element from human protoplast fusion, calcium phosphate precipitation, cell cytomegalovirus is an effective expression system for anti fusion with enveloped DNA, microinjection, and infection bodies (Foecking et al., Gene 45:101 (1986); Cockett et al., with intact virus. See, Ridgway, A. A. G. "Mammalian Bio/Technology 8:2 (1990)). Expression Vectors' Vectors, Rodriguez, and Denhardt, Eds. 65 The host cell line used for protein expression is often of Butterworths, Boston, Mass., Chapter 24.2, pp. 470-472 mammalian origin; those skilled in the art are credited with (1988). Typically, plasmid introduction into the host is via ability to preferentially determine particular host cell lines US 8,551,476 B2 87 88 which are best suited for the desired gene product to be art of recombinant DNA technology which can be used are expressed therein. Exemplary host cell lines include, but are described in Ausubel et al. (eds.), Current Protocols in not limited to, CHO (Chinese Hamster Ovary), DG44 and Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, DUXB11 (Chinese Hamster Ovary lines, DHFR minus), Gene Transfer and Expression, A Laboratory Manual, Stock HELA (human cervical carcinoma). CVI (monkey kidney ton Press, NY (1990); and in Chapters 12 and 13, Dracopoliet line), COS (a derivative of CVI with SV40 Tantigen), VERY. al. (eds), Current Protocols in Human Genetics, John BHK (baby hamster kidney), MDCK, 293, WI38, R1610 Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. (Chinese hamster fibroblast) BALBC/3T3 (mouse fibro Biol. 150:1 (1981), which are incorporated by reference blast), HAK (hamster kidney line), SP2/O (mouse myeloma), herein in their entireties. P3x63-Ag3.653 (mouse myeloma), BFA-1c1 BPT (bovine 10 The expression levels of an antibody molecule can be endothelial cells), RAJI (human lymphocyte) and 293 (hu increased by vector amplification (for a review, see Bebbing man kidney). CHO cells are particularly preferred. Host cell ton and Hentschel. The use of vectors based on gene ampli lines are typically available from commercial services, the fication for the expression of cloned genes in mammalian cells American Tissue Culture Collection or from published litera in DNA cloning, Academic Press, New York, Vol. 3. (1987)). ture. 15 When a marker in the vector system expressing antibody is In addition, a host cell Strain may be chosen which modu amplifiable, increase in the level of inhibitor present in culture lates the expression of the inserted sequences, or modifies and of host cell will increase the number of copies of the marker processes the gene product in the specific fashion desired. gene. Since the amplified region is associated with the anti Such modifications (e.g., glycosylation) and processing (e.g., body gene, production of the antibody will also increase cleavage) of protein products may be important for the func (Crouse et al., Mol. Cell. Biol. 3:257 (1983)). tion of the protein. Different host cells have characteristic and In vitro productionallows scale-up to give large amounts of specific mechanisms for the post-translational processing and the desired polypeptides. Techniques for mammalian cell modification of proteins and gene products. Appropriate cell cultivation under tissue culture conditions are known in the lines or host systems can be chosen to ensure the correct art and include homogeneous Suspension culture, e.g. in an modification and processing of the foreign protein expressed. 25 airlift reactor or in a continuous stirrer reactor, or immobi To this end, eukaryotic host cells which possess the cellular lized or entrapped cell culture, e.g. in hollow fibers, micro machinery for proper processing of the primary transcript, capsules, on agarose microbeads or ceramic cartridges. If glycosylation, and phosphorylation of the gene product may necessary and/or desired, the Solutions of polypeptides can be be used. purified by the customary chromatography methods, for For long-term, high-yield production of recombinant pro 30 example gel filtration, ion-exchange chromatography, chro teins, stable expression is preferred. For example, cell lines matography over DEAE-cellulose or (immuno-)affinity chro which stably express the antibody molecule may be engi matography, e.g., after preferential biosynthesis of a synthetic neered. Rather than using expression vectors which contain hinge region polypeptide or prior to or Subsequent to the HIC viral origins of replication, host cells can be transformed with chromatography step described herein. DNA controlled by appropriate expression control elements 35 Genes encoding Sp35 antibodies, or antigen-binding frag (e.g., promoter, enhancer, sequences, transcription termina ments, variants, or derivatives thereof of the invention can tors, polyadenylation sites, etc.), and a selectable marker. also be expressed non-mammalian cells such as bacteria or Following the introduction of the foreign DNA, engineered yeast or plant cells. Bacteria which readily take up nucleic cells may be allowed to grow for 1-2 days in an enriched acids include members of the enterobacteriaceae. Such as media, and then are switched to a selective media. The select 40 strains of Escherichia coli or Salmonella; Bacillaceae, such able marker in the recombinant plasmid confers resistance to as Bacillus subtilis, Pneumococcus, Streptococcus, and Hae the selection and allows cells to stably integrate the plasmid mophilus influenzae. It will further be appreciated that, when into their chromosomes and grow to form foci which in turn expressed in bacteria, the heterologous polypeptides typi can be cloned and expanded into cell lines. This method may cally become part of inclusion bodies. The heterologous advantageously be used to engineer cell lines which stably 45 polypeptides must be isolated, purified and then assembled express the antibody molecule. into functional molecules. Where tetravalent forms of anti A number of selection systems may be used, including but bodies are desired, the subunits will then self-assemble into not limited to the herpes simplex virus thymidine kinase tetravalent antibodies (WO02/096948A2). (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine In bacterial systems, a number of expression vectors may phosphoribosyltransferase (Szybalska & Szybalski, Proc. 50 be advantageously selected depending upon the use intended Natl. Acad. Sci. USA 48:202 (1992)), and adenine phospho for the antibody molecule being expressed. For example, ribosyltransferase (Lowy et al., Cell 22:817 1980) genes can when a large quantity of such a protein is to be produced, for be employed in tha-, hgprt- or aprt-cells, respectively. Also, the generation of pharmaceutical compositions of an anti antimetabolite resistance can be used as the basis of selection body molecule, vectors which direct the expression of high for the following genes: dhfr, which confers resistance to 55 levels of fusion protein products that are readily purified may methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 be desirable. Such vectors include, but are not limited, to the (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 E. coli expression vector puR278 (Ruther et al., EMBO.J. (1981)); gpt, which confers resistance to mycophenolic acid 2:1791 (1983)), in which the antibody coding sequence may (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 be ligated individually into the vector in frame with the lacz (1981)); neo, which confers resistance to the aminoglycoside 60 coding region so that a fusion protein is produced; plN vec G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Bio tors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 therapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503–5509 Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1989)); and the like. pGEX vectors may also be used to (1993); and Morgan and Anderson, Ann. Rev. Biochem. express foreign polypeptides as fusion proteins with glu 62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); 65 tathione S-transferase (GST). In general, such fusion proteins and hygro, which confers resistance to hygromycin (Santerre are soluble and can easily be purified from lysed cells by et al., Gene 30:147 (1984). Methods commonly known in the adsorption and binding to a matrix glutathione-agarose beads US 8,551,476 B2 89 90 followed by elution in the presence of free glutathione. The metachromatic leukodystrophy, trigeminal neuralgia, Bell's pGEX vectors are designed to include thrombin or factor Xa palsy, spinal cord injury and all neurological diseases related protease cleavage sites so that the cloned target gene product to neuronal cell death. can be released from the GST moiety. The inventors have further discovered that Sp35 is In addition to prokaryotes, eukaryotic microbes may also expressed in oligodendrocytes, and contributes to oligoden be used. Saccharomyces cerevisiae, or common baker's drocyte biology. Soluble derivatives of Sp35, certain poly yeast, is the most commonly used among eukaryotic micro nucleotides (e.g. RNAi), as well as certain antibodies which organisms although a number of other strains are commonly specifically bind to Sp35, as described herein act as antago available, e.g., Pichia pastoris. nists to Sp35 function in oligodendrocytes, promoting prolif For expression in Saccharomyces, the plasmid YRp7, for 10 eration, differentiation and Survival of oligodendrocytes and promoting myelination of neurons in vitro and in vivo. This is example, (Stinchcomb et al., Nature 282:39 (1979); Kings beneficial in for diseases, disorders or conditions involving man et al., Gene 7:141 (1979); Tschemperet al., Gene 10:157 demyelination and dysmyelination. Examples of diseases or (1980)) is commonly used. This plasmid already contains the disorders in which oligodendrocyte proliferation, differentia TRP1 gene which provides a selection marker for a mutant 15 tion and Survival, and/or myelination or remyelination would strain of yeast lacking the ability to grow in tryptophan, for be beneficial include multiple sclerosis (MS), progressive example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 multifocal leukoencephalopathy (PML), encephalomyelitis (1977)). The presence of the trp 1 lesion as a characteristic of (EPL), central pontine myelolysis (CPM), adrenoleukodys the yeast host cell genome then provides an effective envi trophy, Alexander's disease, Pelizaeus Merzbacher disease ronment for detecting transformation by growth in the (PMZ), Globoid cell Leucodystrophy (Krabbe's disease), absence of tryptophan. Wallerian Degeneration, optic neuritis, transverse myelitis, In an insect system, Autographa Californica nuclear poly amylotrophic lateral Sclerosis (ALS), Huntington's disease, hedrosis virus (AcNPV) is typically used as a vector to Alzheimer's disease, Parkinson's disease, spinal cord injury, express foreign genes. The virus grows in Spodoptera fru traumatic brain injury, post radiation injury, neurologic com giperda cells. The antibody coding sequence may be cloned 25 plications of chemotherapy, stroke, acute ischemic optic neu individually into non-essential regions (for example the poly ropathy, vitamin E deficiency, isolated vitamin E deficiency hedrin gene) of the virus and placed under control of an syndrome, AR, Bassen-KornZweig syndrome, Marchiafava AcNPV promoter (for example the polyhedrin promoter). Bignami syndrome, metachromatic leukodystrophy, trigemi Once an antibody molecule of the invention has been nal neuralgia, and Bell's palsy. recombinantly expressed, it may be purified by any method 30 Accordingly, one embodiment of the present invention known in the art for purification of an immunoglobulin mol provides methods for treating spinal cord injury, diseases or ecule, for example, by chromatography (e.g., ion exchange, disorders associated with inhibition of neuronal growth in the affinity, particularly by affinity for the specific antigen after CNS, diseases or disorders associated with inhibition of oli Protein A, and sizing column chromatography), centrifuga godendrocyte growth or differentiation, and diseases involv tion, differential solubility, or by any other standard technique 35 ing demyelination or dysmyelination of CNS neurons in an for the purification of proteins. Alternatively, a preferred animal Suffering from Such injury or disease or predisposed to method for increasing the affinity of antibodies of the inven contract Such disease, the method comprising, consisting tion is disclosed in US 2002 0123057 A1. essentially of, or consisting of administering to the animal an effective amount of an Sp35 antibody, or antigen-binding VIII. Treatment Methods. Using Therapeutic Sp35 40 fragment, variant, or derivative thereof. Antibodies of the Antibodies invention are described herein, and include the monoclonal antibodies listed in Table 3A and 3B, antibodies which spe As described herein, Sp35 antibodies, or antigen-binding cifically bind to the same epitope as the monoclonal antibod fragments, variants, orderivatives thereof of the invention can ies listed in Table 3A and 3B, antibodies which competitively relieve NgR1-mediated inhibition of axonal extension that 45 inhibit binding of the monoclonal antibodies listed in Table normally takes place in CNS neurons. This is beneficial in 3A and 3B to Sp35, and antibodies comprising polypeptides situations where axonal extension or neurite sprouting is derived from the monoclonal antibodies listed in Table 3A needed in the brain or spinal cord. Spinal cord injury, includ and 3B. ing partial or complete crush or severance, exemplifies a A therapeutic Sp35 antibody to be used in treatment meth situation in which axonal extension is needed, but is normally 50 ods disclosed hereincan be prepared and used as a therapeutic inhibited through operation of the Nogo pathway. Examples agent which promotes CNS neurite outgrowth, neuronal Sur of diseases or disorders in which axonal extension and/or vival, axon guidance and axon regeneration, which promotes neurite sprouting in the brain would be beneficial include oligodendrocyte Survival, growth, and/or differentiation, and stroke, multiple Sclerosis, and other neurodegenerative dis which promotes myelination or remyelination of CNS neu eases or disorders such as multiple Sclerosis (MS), progres 55 rons. Characteristics of suitable therapeutic Sp35 antibodies sive multifocal leukoencephalopathy (PML), encephalomy include: binding to Sp35 epitopes which result in blocking of elitis (EPL), central pontine myelolysis (CPM), Sp35 activity, binding to Sp35 with sufficient affinity to elicit adrenoleukodystrophy, Alexander's disease, Pelizaeus Merz a therapeutic effect, and binding to Sp35 preferentially to bacher disease (PMZ), Globoid cell Leucodystrophy normal binding partners, e.g., Nogo Receptor. (Krabbe's disease) and Wallerian Degeneration, optic neuri 60 Therapeutic Sp35 antibodies may be monoclonal, chimeric tis, transverse myelitis, amylotrophic lateral Sclerosis (ALS), or humanized antibodies, or fragments of antibodies that bind Huntington's disease, Alzheimer's disease, Parkinson's dis specifically to Sp35. The antibodies may be monovalent, ease, spinal cord injury, traumatic brain injury, post radiation bivalent, polyvalent, or bifunctional antibodies. Antibody injury, neurologic complications of chemotherapy, stroke, fragments include without limitation Fab F(ab'), and Fv neuropathy, acute ischemic optic neuropathy, vitamin E defi 65 fragments. ciency, isolated vitamin E deficiency syndrome, AR, Bassen Therapeutic Sp35 antibodies, or antigen-binding frag Kornzweig syndrome, Marchiafava-Bignami syndrome, ments, variants or derivatives thereof according to the inven US 8,551,476 B2 91 92 tion can be used in unlabeled or unconjugated form, or can be The methods for treating spinal cord injury, diseases or coupled or linked to drugs, labels or stabilization agents disorders associated with inhibition of neuronal growth in the which may or may not exert additional therapeutic effects. CNS, diseases or disorders associated with inhibition of oli A specific dosage and treatment regimen for any particular godendrocyte growth or differentiation, and diseases involv patient will depend upon a variety of factors, including the ing demyelination or dysmyelination of CNS neurons com particular Sp35 antibody, or antigen-binding fragment, Vari prising administration of an Sp35 antibody, or antigen ant or derivative thereofused, the patient’s age, body weight, binding fragment, variant, or derivative thereof of the general health, sex, and diet, and the time of administration, invention are typically tested in vitro, and then in vivo in an rate of excretion, drug combination, and the severity of the acceptable animal model, for the desired therapeutic or pro particular disease being treated. Judgment of Such factors by 10 phylactic activity, prior to use in humans. Suitable animal models, including transgenic animals, are will knownto those medical caregivers is within the ordinary skill in the art. The of ordinary skill in the art. For example, in vitro assays to amount will also depend on the individual patient to be demonstrate the therapeutic utility of Sp35 antibody treated, the route of administration, the type of formulation, described herein include the effect of an Sp35 antibody on a the characteristics of the compound used, the severity of the 15 cell line or a patient tissue sample. The effect of the Sp35 disease, and the desired effect. The amount used can be deter antibody on the cell line and/or tissue sample can be deter mined by pharmacological and pharmacokinetic principles mined utilizing techniques known to those of skill in the art, well known in the art. Such as the assays disclosed elsewhere herein. In accordance In the methods of the invention the Sp35 antibodies, or with the invention, in vitro assays which can be used to antigen-binding fragments, variants or derivatives thereof determine whether administration of a specific Sp35 antibody may be administered directly to the nervous system, intrac is indicated, include in vitro cell culture assays in which a erebroVentricularly, or intrathecally, e.g. into a chroniclesion patient tissue sample is grown in culture, and exposed to or of MS, as discussed in more detail below. otherwise administered a compound, and the effect of Such In various embodiments, an Sp35 antibody as described compound upon the tissue sample is observed. above is an antagonist of Sp35 activity. In certain embodi 25 Supplementary active compounds also can be incorporated ments, for example, binding of an antagonist Sp35 antibody into the compositions of the invention. For example, a Sp35 to Sp35, as expressed on neurons, blocks myelin-associated antibody, or antigen-binding fragment, variant, or derivative neurite outgrowth inhibition or neuronal cell death. In other thereof of the invention may be coformulated with and/or embodiments, binding of the Sp35 antibody to Sp35, as coadministered with one or more additional therapeutic expressed on oligodendrocytes, blocks inhibition of oligo 30 agents. dendrocyte growth or differentiation, or blocks demyelina The invention encompasses any Suitable delivery method tion or dysmyelination of CNS neurons. for a Sp35 antibody, or antigen-binding fragment, variant, or In methods of the present invention, an Sp35 antibody, or derivative thereof of the invention to a selected target tissue, an antigen-binding fragment, variant, or derivative thereof, in including bolus injection of an aqueous solution or implan particular the Sp35 antibodies described herein, can be 35 tation of a controlled-release system. Use of a controlled administered directly as a preformed polypeptide, or indi release implant reduces the need for repeat injections. rectly through a nucleic acid vector, to permit beneficial axonal outgrowth, promote oligodendrocyte proliferation, IX. Pharmaceutical Compositions and differentiation, and Survival, and/or promote myelination or Administration Methods remyelination. 40 In certain embodiments, a Subject may be treated with a Methods of preparing and administering Sp35 antibodies, nucleic acid molecule encoding an Sp35 antibody, orantigen or antigen-binding fragments, variants, or derivatives thereof binding fragment, variant, or analog thereof, e.g., in a vector. of the invention to a subject in need thereofare well known to Doses for nucleic acids encoding polypeptides range from or are readily determined by those skilled in the art. The route about 10 ng to 1 g, 100ng to 100 mg, 1 lug to 10 mg. or 30-300 45 of administration of the Sp35 antibody, or antigen-binding ug DNA per patient. Doses for infectious viral vectors vary fragment, variant, or derivative thereof may be, for example, from 10-100, or more, virions per dose. oral, parenteral, by inhalation or topical. The term parenteral In some embodiments of the present invention an Sp35 as used herein includes, e.g., intravenous, intraarterial, intra antibody, or an antigen-binding fragment, variant, or deriva peritoneal, intramuscular, Subcutaneous, rectal or vaginal tive thereof is administered in a treatment method that 50 administration. While all these forms of administration are includes: (1) transforming ortransfecting an implantable host clearly contemplated as being within the Scope of the inven cell with a nucleic acid, e.g., a vector, that expresses an Sp35 tion, a form for administration would be a solution for injec antibody, or an antigen-binding fragment, variant, or deriva tion, in particular for intravenous or intraarterial injection or tive thereof; and (2) implanting the transformed host cell into drip. Usually, a suitable pharmaceutical composition for a mammal, at the site of a disease, disorder or injury. For 55 injection may comprise a buffer (e.g. acetate, phosphate or example, the transformed host cell can be implanted at the site citrate buffer), a Surfactant (e.g. polysorbate), optionally a of a spinal cord injury or at a site of dysmyelination. In some stabilizer agent (e.g. human albumin), etc. However, in other embodiments of the invention, the implantable host cell is methods compatible with the teachings herein, Sp35 antibod removed from a mammal, temporarily cultured, transformed ies, or antigen-binding fragments, variants, or derivatives or transfected with an isolated nucleic acid encoding a an 60 thereof of the invention can be delivered directly to the site of Sp35 antibody, and implanted back into the same mammal the adverse cellular population thereby increasing the expo from which it was removed. The cell can be, but is not Sure of the diseased tissue to the therapeutic agent. required to be, removed from the same site at which it is As previously discussed, Sp35 antibodies, orantigen-bind implanted. Such embodiments, sometimes known as ex vivo ing fragments, variants, or derivatives thereof of the invention gene therapy, can provide a continuous Supply of the Sp35 65 may be administered in a pharmaceutically effective amount polypeptide, localized at the site of site of action, for a limited for the in vivo treatment of mammalian spinal cord injury, period of time. diseases or disorders associated with inhibition of neuronal US 8,551,476 B2 93 94 growth in the CNS, diseases or disorders associated with example, parabens, chlorobutanol, phenol, ascorbic acid, inhibition of oligodendrocyte growth or differentiation, and thimerosal and the like. In many cases, it will be preferable to diseases involving demyelination or dysmyelination of CNS. include isotonic agents, for example, Sugars, polyalcohols, In this regard, it will be appreciated that the disclosed anti Such as mannitol, Sorbitol, or sodium chloride in the compo bodies will be formulated so as to facilitate administration sition. Prolonged absorption of the injectable compositions and promote stability of the active agent. Preferably, pharma can be brought about by including in the composition an agent ceutical compositions in accordance with the present inven which delays absorption, for example, aluminum monostear tion comprise a pharmaceutically acceptable, non-toxic, ster ate and gelatin. ile carrier Such as physiological saline, non-toxic buffers, In any case, Sterile injectable solutions can be prepared by preservatives and the like. For the purposes of the instant 10 incorporating an active compound (e.g., an Sp35 antibody, or application, a pharmaceutically effective amount of an Sp35 antigen-binding fragment, variant, or derivative thereof, by antibody, or antigen-binding fragment, variant, or derivative itself or in combination with other active agents) in the thereof, conjugated or unconjugated, shall be held to mean an required amount in an appropriate solvent with one or a amount Sufficient to achieve effective binding to a target and combination of ingredients enumerated herein, as required, to achieve a benefit, e.g., to ameliorate symptoms of a disease 15 followed by filtered sterilization. Generally, dispersions are or disorder or to detect a substance or a cell. prepared by incorporating the active compound into a sterile The pharmaceutical compositions used in this invention vehicle, which contains a basic dispersion medium and the comprise pharmaceutically acceptable carriers, including, required other ingredients from those enumerated above. In e.g., ion exchangers, alumina, aluminum Stearate, lecithin, the case of sterile powders for the preparation of sterile inject serum proteins, such as human serum albumin, buffer Sub able solutions, the preferred methods of preparation are stances such as phosphates, glycine, Sorbic acid, potassium vacuum drying and freeze-drying, which yields a powder of Sorbate, partial glyceride mixtures of Saturated vegetable an active ingredient plus any additional desired ingredient fatty acids, water, salts or electrolytes, such as protamine from a previously sterile-filtered solution thereof. The prepa Sulfate, disodium hydrogen phosphate, potassium hydrogen rations for injections are processed, filled into containers such phosphate, Sodium chloride, Zinc salts, colloidal silica, mag 25 as ampoules, bags, bottles, Syringes or vials, and sealed under nesium trisilicate, polyvinyl pyrrolidone, cellulose-based aseptic conditions according to methods known in the art. Substances, polyethylene glycol, Sodium carboxymethylcel Further, the preparations may be packaged and sold in the lulose, polyacrylates, waxes, polyethylene-polyoxypropy form of a kit such as those described in co-pending U.S. Ser. lene-block polymers, polyethylene glycol and wool fat. No. 09/259,337 (US-2002-0102208 A1), which is incorpo Preparations for parenteral administration includes sterile 30 rated herein by reference in its entirety. Such articles of manu aqueous or non-aqueous solutions, Suspensions, and emul facture will preferably have labels or package inserts indicat sions. Examples of non-aqueous solvents are propylene gly ing that the associated compositions are useful for treating a col, polyethylene glycol, vegetable oils such as olive oil, and Subject Suffering from, or predisposed to autoimmune or neo injectable organic esters such as ethyl oleate. Aqueous carri plastic disorders. ers include water, alcoholic/aqueous solutions, emulsions or 35 Parenteral formulations may be a single bolus dose, an Suspensions, including saline and buffered media. In the Sub infusion or a loading bolus dose followed with a maintenance ject invention, pharmaceutically acceptable carriers include, dose. These compositions may be administered at specific but are not limited to, 0.01-0.1M and preferably 0.05M phos fixed or variable intervals, e.g., once a day, or on an “as phate buffer or 0.8% saline. Other common parenteral needed' basis. vehicles include Sodium phosphate solutions, Ringer's dex 40 Certain pharmaceutical compositions used in this inven trose, dextrose and Sodium chloride, lactated Ringers, or tion may be orally administered in an acceptable dosage form fixed oils. Intravenous vehicles include fluid and nutrient including, e.g., capsules, tablets, aqueous Suspensions or replenishers, electrolyte replenishers, such as those based on Solutions. Certain pharmaceutical compositions also may be Ringer's dextrose, and the like. Preservatives and other addi administered by nasal aerosol or inhalation. Such composi tives may also be present Such as for example, antimicrobials, 45 tions may be prepared as solutions in Saline, employing ben antioxidants, chelating agents, and inert gases and the like. Zyl alcohol or other suitable preservatives, absorption pro More particularly, pharmaceutical compositions suitable moters to enhance bioavailability, and/or other conventional for injectable use include sterile aqueous Solutions (where solubilizing or dispersing agents. water soluble) or dispersions and sterile powders for the The amount of an Sp35 antibody, or fragment, variant, or extemporaneous preparation of sterile injectable solutions or 50 derivative thereof that may be combined with the carrier dispersions. In Such cases, the composition must be sterile materials to produce a single dosage form will vary depend and should be fluid to the extent that easy syringability exists. ing upon the host treated and the particular mode of admin It should be stable under the conditions of manufacture and istration. The composition may be administered as a single storage and will preferably be preserved against the contami dose, multiple doses or over an established period of time in nating action of microorganisms, such as bacteria and fungi. 55 an infusion. Dosage regimens also may be adjusted to provide The carrier can be a solvent or dispersion medium containing, the optimum desired response (e.g., a therapeutic or prophy for example, water, ethanol, polyol (e.g., glycerol, propylene lactic response). glycol, and liquid polyethylene glycol, and the like), and In keeping with the scope of the present disclosure, Sp35 suitable mixtures thereof. The proper fluidity can be main antibodies, or antigen-binding fragments, variants, or deriva tained, for example, by the use of a coating Such as lecithin, by 60 tives thereof of the invention may be administered to a human the maintenance of the required particle size in the case of or other animal in accordance with the aforementioned meth dispersion and by the use of surfactants. Suitable formula ods of treatment in an amount Sufficient to produce a thera tions for use in the therapeutic methods disclosed herein are peutic effect. The Sp35 antibodies, or antigen-binding frag described in Remington’s Pharmaceutical Sciences, Mack ments, variants, or derivatives thereof of the invention can be Publishing Co., 16th ed. (1980). 65 administered to Such human or other animal in a conventional Prevention of the action of microorganisms can be dosage form prepared by combining the antibody of the achieved by various antibacterial and antifungal agents, for invention with a conventional pharmaceutically acceptable US 8,551,476 B2 95 96 carrier or diluent according to known techniques. It will be In one embodiment, the Sp35 antibodies, or antigen-binding recognized by one of skill in the art that the form and char fragments, variants, orderivatives thereof of the invention can acter of the pharmaceutically acceptable carrier or diluent is be administered in unconjugated form. In another embodi dictated by the amount of active ingredient with which it is to ment, the Sp35 antibodies, or antigen-binding fragments, be combined, the route of administration and other well 5 variants, or derivatives thereof of the invention can be admin known variables. Those skilled in the art will further appre istered multiple times in conjugated form. In still another ciate that a cocktail comprising one or more species of Sp35 embodiment, Sp35 antibodies, orantigen-binding fragments, antibodies, orantigen-binding fragments, variants, or deriva variants, or derivatives thereof of the invention can be admin tives thereof of the invention may prove to be particularly istered in unconjugated form, then in conjugated form, or vice effective. 10 WSa. Effective doses of the compositions of the present inven The compositions of the present invention may be admin tion, for treatment of spinal cord injury, diseases or disorders istered by any Suitable method, e.g., parenterally, intraven associated with inhibition of neuronal growth in the CNS, tricularly, orally, by inhalation spray, topically, rectally, diseases or disorders associated with inhibition of oligoden nasally, buccally, vaginally or via an implanted reservoir. The drocyte growth or differentiation, and diseases involving 15 term "parenteral” as used herein includes Subcutaneous, demyelination or dysmyelination of CNS vary depending intravenous, intramuscular, intra-articular, intra-synovial, upon many different factors, including means of administra intrasternal, intrathecal, intrahepatic, intralesional and intrac tion, target site, physiological state of the patient, whether the ranial injection or infusion techniques. As described previ patient is human or an animal, other medications adminis ously, Sp35 antibodies, or antigen-binding fragments, vari tered, and whether treatment is prophylactic or therapeutic. ants, or derivatives thereof of the invention act in the nervous Usually, the patient is a human but non-human mammals system to promote survival, proliferation and differentiation including transgenic mammals can also be treated. Treatment of oligodendrocytes and myelination of neurons and neuronal dosages may be titrated using routine methods knownto those Survival, axon regeneration and axon guidance. Accordingly, of skill in the art to optimize safety and efficacy. in the methods of the invention, the Sp35 antibodies, or anti For treatment of spinal cord injury, diseases or disorders 25 gen-binding fragments, variants, or derivatives thereof are associated with inhibition of neuronal growth in the CNS, administered in such a way that they cross the blood-brain diseases or disorders associated with inhibition of oligoden barrier. This crossing can result from the physico-chemical drocyte growth or differentiation, and diseases involving properties inherent in the Sp35 antibody molecule itself, from demyelination or dysmyelination of CNS with an Sp35 anti other components in a pharmaceutical formulation, or from body, or antigen-binding fragment, variant, or derivative 30 the use of a mechanical device Such as a needle, cannula or thereof, the dosage can range, e.g., from about 0.0001 to 100 surgical instruments to breach the blood-brain barrier. Where mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg. the Sp35 antibody is a molecule that does not inherently cross 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.). the blood-brain barrier, e.g., a fusion to a moiety that facili of the host body weight. For example dosages can be 1 mg/kg tates the crossing, Suitable routes of administration are, e.g., body weight or 10 mg/kg body weight or within the range of 35 intrathecal or intracranial, e.g., directly into a chronic lesion 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate of MS. Where the Sp35 antibody is a molecule that inherently in the above ranges are also intended to be within the scope of crosses the blood-brain barrier, the route of administration the invention. Subjects can be administered such doses daily, may be by one or more of the various routes described below. on alternative days, weekly or according to any other sched In some methods, antibodies are administered as a Sustained ule determined by empirical analysis. An exemplary treat 40 release composition or device, such as a MedipadTM device. ment entails administration in multiple dosages over a pro Delivery across the blood brain barrier can be enhanced by a longed period, for example, of at least six months. Additional carrying molecule. Such as anti-Fc receptor, transferrin, anti exemplary treatment regimes entail administration once per insulin receptor or a toxin conjugate or penetration enhancer. every two weeks or once a month or once every 3 to 6 months. The Sp35 antibodies, or antigen-binding fragments, vari Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg 45 ants, or derivatives thereofused in the methods of the inven on consecutive days, 30 mg/kg on alternate days or 60 mg/kg tion may be directly infused into the brain. Various implants weekly. In some methods, two or more monoclonal antibod for direct brain infusion of compounds are known and are ies with different binding specificities are administered effective in the delivery of therapeutic compounds to human simultaneously, in which case the dosage of each antibody patients suffering from neurological disorders. These include administered falls within the ranges indicated. 50 chronic infusion into the brain using a pump, Stereotactically Sp35 antibodies, orantigen-binding fragments, variants, or implanted, temporary interstitial catheters, permanent intrac derivatives thereof of the invention can be administered on ranial catheter implants, and Surgically implanted biodegrad multiple occasions. Intervals between single dosages can be able implants. See, e.g., Gill et al., “Direct brain infusion of daily, weekly, monthly or yearly. Intervals can also be irregu glial cell line-derived neurotrophic factor in Parkinson dis lar as indicated by measuring blood levels of target polypep 55 ease.” Nature Med. 9:589-95 (2003); Scharfen et al., “High tide or target molecule in the patient. In some methods, dos Activity Iodine-125 Interstitial Implant For Gliomas.” Int. J. age is adjusted to achieve a plasma polypeptide concentration Radiation Oncology Biol. Phys. 24(4):583-91 (1992); Gaspar of 1-1000 ug/ml and in some methods 25-300 ug/ml. Alter et al., “Permanent 'I Implants for Recurrent Malignant natively, Sp35 antibodies, orantigen-binding fragments, Vari Gliomas.” Int. J. Radiation Oncology Biol. Phys. 43(5):977 ants, or derivatives thereof of the invention can be adminis 60 82 (1999); chapter 66, pages 577-580, Bellezza et al., “Ster tered as a Sustained release formulation, in which case less eotactic Interstitial Brachytherapy,” in Gildenberget al., Text frequent administration is required. Dosage and frequency book of Stereotactic and Functional Neurosurgery, McGraw vary depending on the half-life of the antibody in the patient. Hill (1998); and Brem et al., “The Safety of Interstitial The half-life of an Sp35 antibody can also be prolonged via Chemotherapy with BCNU-Loaded Polymer Followed by fusion to a stable polypeptide or moeity, e.g., albuminor PEG. 65 Radiation Therapy in the Treatment of Newly Diagnosed In general, humanized antibodies show the longest half-life, Malignant Gliomas: Phase I Trial. J. Neuro-Oncology followed by chimeric antibodies and nonhuman antibodies. 26:111-23 (1995). US 8,551,476 B2 97 98 The compositions may also comprise an Sp35 antibody assay (ELISA), immunoprecipitation, or western blotting. dispersed in a biocompatible carrier material that functions as Suitable assays are described in more detail elsewhere herein. a suitable delivery or Support system for the compounds. By “assaying the expression level of Sp35 polypeptide' is Suitable examples of Sustained release carriers include semi intended qualitatively or quantitatively measuring or estimat permeable polymer matrices in the form of shaped articles ing the level of Sp35 polypeptide in a first biological sample Such as Suppositories or capsules. Implantable or microcap either directly (e.g., by determining or estimating absolute Sular Sustained release matrices include polylactides (U.S. protein level) or relatively (e.g., by comparing to the cancer Pat. No. 3,773,319; EP 58.481), copolymers of L-glutamic associated polypeptide level in a second biological sample). acid and gamma-ethyl-L-glutamate (Sidman et al., Biopoly Preferably, Sp35 polypeptide expression level in the first bio mers 22:547-56 (1985)); poly(2-hydroxyethyl-methacry 10 logical sample is measured or estimated and compared to a late), ethylene vinyl acetate (Langer et al., J. Biomed. Mater. standard Sp35 polypeptide level, the standard being taken Res. 15:167-277 (1981); Langer, Chem. Tech. 12:98-105 from a second biological sample obtained from an individual (1982)) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). not having the disorder or being determined by averaging In some embodiments of the invention, an Sp35 antibody, 15 levels from a population of individuals not having the disor or antigen-binding fragment, variant, or derivative thereof of der. As will be appreciated in the art, once the “standard the invention is administered to a patient by direct infusion Sp35 polypeptide level is known, it can be used repeatedly as into an appropriate region of the brain. See, e.g., Gill et al., a standard for comparison. Supra. Alternative techniques are available and may be By “biological sample' is intended any biological sample applied to administer an Sp35 antibody according to the obtained from an individual, cell line, tissue culture, or other invention. For example, Stereotactic placement of a catheter source of cells potentially expressing Sp35. Methods for or implant can be accomplished using the Riechert-Mund obtaining tissue biopsies and body fluids from mammals are inger unit and the ZD (Zamorano-Dujovny) multipurpose well known in the art. localizing unit. A contrast-enhanced computerized tomogra Sp35 antibodies for use in the diagnostic methods phy (CT) scan, injecting 120 ml of omnipaque, 350 mg 25 described above include any Sp35 antibody which specifi iodine/ml, with 2 mm slice thickness can allow three-dimen cally binds to an Sp35 gene product, as described elsewhere sional multiplanar treatment planning (STP. Fischer, herein. Freiburg, Germany). This equipment permits planning on the basis of magnetic resonance imaging studies, merging the CT XI. Immunoassays and MRI target information for clear target confirmation. 30 The Leksell stereotactic system (Downs Surgical, Inc., Sp35 antibodies, orantigen-binding fragments, variants, or Decatur, Ga.) modified for use with a GE CT scanner (Gen derivatives thereof of the invention may be assayed for immu eral Electric Company, Milwaukee, Wis.) as well as the nospecific binding by any method known in the art. The Brown-Roberts-Wells (BRW) stereotactic system (Radion immunoassays which can be used include but are not limited ics, Burlington, Mass.) can be used for this purpose. Thus, on 35 to competitive and non-competitive assay systems using tech the morning of the implant, the annular base ring of the BRW niques such as western blots, radioimmunoassays, ELISA stereotactic frame can be attached to the patient’s skull. Serial (enzyme linked immunosorbent assay), 'sandwich’ immu CT sections can be obtained at 3 mm intervals though the noassays, immunoprecipitation assays, precipitin reactions, (target tissue) region with a graphite rod localizer frame gel diffusion precipitin reactions, immunodiffusion assays, clamped to the base plate. A computerized treatment planning 40 agglutination assays, complement-fixation assays, immuno program can be run on a VAX 11/780 computer (Digital radiometric assays, fluorescent immunoassays, protein A Equipment Corporation, Maynard, Mass.) using CT coordi immunoassays, to name but a few. Such assays are routine and nates of the graphite rod images to map between CT space and well known in the art (see, e.g., Ausubel et al., eds, Current BRW space. Protocols in Molecular Biology, John Wiley & Sons, Inc., Sp35 antibodies, orantigen-binding fragments, variants, or 45 New York, Vol. 1 (1994), which is incorporated by reference derivatives thereof of the invention can optionally be admin herein in its entirety). Exemplary immunoassays are istered in combination with other agents that are effective in described briefly below (but are not intended by way of limi treating the disorder or condition in need of treatment (e.g., tation). prophylactic or therapeutic). Immunoprecipitation protocols generally comprise lysing 50 a population of cells in a lysis buffer such as RIPA buffer (1% X. Diagnostics NP-40 or TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Tra The invention further provides a diagnostic method useful sylol) supplemented with protein phosphatase and/or pro during diagnosis of neuronal disorders or injuries, which tease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vana involves measuring the expression level of Sp35 protein or 55 date), adding the antibody of interest to the cell lysate, transcript in tissue or other cells or body fluid from an indi incubating for a period of time (e.g., 1-4 hours) at 4.degree. vidual and comparing the measured expression level with a C., adding protein A and/or protein G Sepharose beads to the standard Sp35 expression levels in normal tissue or body cell lysate, incubating for about an hour or more at 4.degree. fluid, whereby an increase in the expression level compared to C., washing the beads in lysis buffer and resuspending the the standard is indicative of a disorder. 60 beads in SDS/sample buffer. The ability of the antibody of Sp35-specificantibodies can be used to assay protein levels interest to immunoprecipitate a particular antigen can be in a biological sample using classical immunohistological assessed by, e.g., western blot analysis. One of skill in the art methods known to those of skill in the art (e.g., see Jalkanen, would be knowledgeable as to the parameters that can be et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. modified to increase the binding of the antibody to an antigen Cell Biol. 105:3087-3096 (1987)). Other antibody-based 65 and decrease the background (e.g., pre-clearing the cell lysate methods useful for detecting protein expression include with Sepharose beads). For further discussion regarding immunoassays, such as the enzyme linked immunosorbent immunoprecipitation protocols see, e.g., Ausubel et al., eds, US 8,551,476 B2 99 100 Current Protocols in Molecular Biology, John Wiley & Sons, patient, and applying thereto a labeled Sp35 antibody, or Inc., New York, Vol. 1 (1994) at 10.16.1. antigen-binding fragment, variant, or derivative thereof, pref Western blot analysis generally comprises preparing pro erably applied by overlaying the labeled antibody (or frag tein samples, electrophoresis of the protein samples in a poly ment) onto a biological sample. Through the use of Such a acrylamide gel (e.g., 8%-20% SDS-PAGE depending on the 5 procedure, it is possible to determine not only the presence of molecular weight of the antigen), transferring the protein Sp35 protein, or conserved variants or peptide fragments, but sample from the polyacrylamide gel to a membrane Such as also its distribution in the examined tissue. Using the present nitrocellulose, PVDF or nylon, blocking the membrane in invention, those of ordinary skill will readily perceive that any blocking solution (e.g., PBS with 3% BSA or non-fat milk), of a wide variety of histological methods (such as staining washing the membrane in washing buffer (e.g., PBS-Tween 10 procedures) can be modified in order to achieve Such in situ 20), blocking the membrane with primary antibody (the anti detection. body of interest) diluted in blocking buffer, washing the Immunoassays and non-immunoassays for Sp35 gene membrane in washing buffer, blocking the membrane with a products or conserved variants or peptide fragments thereof secondary antibody (which recognizes the primary antibody, will typically comprise incubating a sample, such as a bio e.g., an anti-human antibody) conjugated to an enzymatic 15 logical fluid, a tissue extract, freshly harvested cells, or Substrate (e.g., horseradish peroxidase or alkaline phos lysates of cells which have been incubated in cell culture, in phatase) or radioactive molecule (e.g., 32p or 1251) diluted in the presence of a detectably labeled antibody capable of bind blocking buffer, washing the membrane in wash buffer, and ing to Sp35 or conserved variants or peptide fragments detecting the presence of the antigen. One of skill in the art thereof, and detecting the bound antibody by any of a number would be knowledgeable as to the parameters that can be of techniques well-known in the art. modified to increase the signal detected and to reduce the The biological sample may be brought in contact with and background noise. For further discussion regarding western immobilized onto a solid phase Support or carrier Such as blot protocols see, e.g., Ausubel et al., eds. Current Protocols nitrocellulose, or other solid support which is capable of in Molecular Biology, John Wiley & Sons, Inc., New York immobilizing cells, cell particles or soluble proteins. The Vol. 1 (1994) at 10.8.1. 25 support may then be washed with suitable buffers followed by ELISAS comprise preparing antigen, coating the well of a treatment with the detectably labeled Sp35 antibody, or anti 96 well microtiter plate with the antigen, adding the antibody gen-binding fragment, variant, or derivative thereof. The of interest conjugated to a detectable compound Such as an solid phase support may then be washed with the buffer a enzymatic Substrate (e.g., horseradish peroxidase or alkaline second time to remove unbound antibody. Optionally the phosphatase) to the well and incubating for a period of time, 30 antibody is subsequently labeled. The amount of bound label and detecting the presence of the antigen. In ELISAS the on Solid Support may then be detected by conventional means. antibody of interest does not have to be conjugated to a By "solid phase support or carrier' is intended any support detectable compound; instead, a second antibody (which rec capable of binding an antigen or an antibody. Well-known ognizes the antibody of interest) conjugated to a detectable Supports or carriers include glass, polystyrene, polypropy compound may be added to the well. Further, instead of 35 lene, polyethylene, dextran, nylon, amylases, natural and coating the well with the antigen, the antibody may be coated modified celluloses, polyacrylamides, gabbros, and magne to the well. In this case, a second antibody conjugated to a tite. The nature of the carrier can be either soluble to some detectable compound may be added following the addition of extent or insoluble for the purposes of the present invention. the antigen of interest to the coated well. One of skill in the art The Support material may have virtually any possible struc would be knowledgeable as to the parameters that can be 40 tural configuration so long as the coupled molecule is capable modified to increase the signal detected as well as other of binding to an antigen or antibody. Thus, the Support con variations of ELISAs known in the art. For further discussion figuration may be spherical, as in a bead, or cylindrical, as in regarding ELISAS see, e.g., Ausubel et al., eds. Current Pro the inside surface of a test tube, or the external surface of a tocols in Molecular Biology, John Wiley & Sons, Inc., New rod. Alternatively, the surface may be flat such as a sheet, test York, Vol. 1 (1994) at 11.2.1. 45 strip, etc. Preferred supports include polystyrene beads. The binding affinity of an antibody to an antigen and the Those skilled in the art will know many other suitable carriers off-rate of an antibody-antigen interaction can be determined for binding antibody orantigen, or will be able to ascertain the by competitive binding assays. One example of a competitive same by use of routine experimentation. binding assay is a radioimmunoassay comprising the incuba The binding activity of a given lot of Sp35 antibody, or tion of labeled antigen (e.g., H or 'I) with the antibody of 50 antigen-binding fragment, variant, or derivative thereof may interest in the presence of increasing amounts of unlabeled be determined according to well known methods. Those antigen, and the detection of the antibody bound to the labeled skilled in the art will be able to determine operative and antigen. The affinity of the antibody of interest for a particular optimal assay conditions for each determination by employ antigen and the binding off-rates can be determined from the ing routine experimentation. data by scatchard plot analysis. Competition with a second 55 There are a variety of methods available for measuring the antibody can also be determined using radioimmunoassays. affinity of an antibody-antigen interaction, but relatively few In this case, the antigen is incubated with antibody of interest for determining rate constants. Most of the methods rely on is conjugated to a labeled compound (e.g., H or 'I) in the either labeling antibody orantigen, which inevitably compli presence of increasing amounts of an unlabeled second anti cates routine measurements and introduces uncertainties in body. 60 the measured quantities. Sp35 antibodies, orantigen-binding fragments, variants, or Surface plasmon reasonance (SPR) as performed on BIA derivatives thereof of the invention, additionally, be core offers a number of advantages over conventional meth employed histologically, as in immunofluorescence, immu ods of measuring the affinity of antibody-antigen interac noelectron microscopy or non-immunological assays, for in tions: (i) no requirement to label either antibody or antigen; situ detection of cancer antigen gene products or conserved 65 (ii) antibodies do not need to be purified in advance, cell variants or peptide fragments thereof. In situ detection may be culture Supernatant can be used directly; (iii) real-time mea accomplished by removing a histological specimen from a Surements, allowing rapid semi-quantitative comparison of US 8,551,476 B2 101 102 different monoclonal antibody interactions, are enabled and binding studies, and can relate functional epitopes to struc are Sufficient for many evaluation purposes; (iv) biospecific tural features when the primary sequence of the antigen is Surface can be regenerated so that a series of different mono known. Peptides orantigen fragments are tested for inhibition clonal antibodies can easily be compared under identical of binding of different MAbs to immobilized antigen. Pep conditions; (V) analytical procedures are fully automated, and 5 tides which interfere with binding of a given MAb are extensive series of measurements can be performed without assumed to be structurally related to the epitope defined by user intervention. BIAapplications Handbook, version AB that MAb. (reprinted 1998), BIACORE code No. BR-1001-86; BIAt The practice of the present invention will employ, unless echnology Handbook, version AB (reprinted 1998), BIA otherwise indicated, conventional techniques of cell biology, CORE code No. BR-1001-84. 10 cell culture, molecular biology, transgenic biology, microbi SPR based binding studies require that one member of a ology, recombinant DNA, and immunology, which are within binding pair be immobilized on a sensor Surface. The binding the skill of the art. Such techniques are explained fully in the partner immobilized is referred to as the ligand. The binding literature. See, for example, Molecular Cloning A Laboratory partner in Solution is referred to as the analyte. In some cases, Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor the ligand is attached indirectly to the Surface through binding 15 Laboratory Press: (1989); Molecular Cloning: A Laboratory to another immobilized molecule, which is referred as the Manual, Sambrook et al., ed., Cold Springs Harbor Labora capturing molecule. SPR response reflects a change in mass tory, New York (1992), DNA Cloning, D. N. Glover ed., concentration at the detector Surface as analytes bind or dis Volumes 1 and 11 (1985); Oligonucleotide Synthesis, M. J. Sociate. Gait ed., (1984); Mullis et al. U.S. Pat. No. 4,683, 195: Based on SPR, real-time BIAcore measurements monitor Nucleic Acid Hybridization, B. D. Hames & S.J. Higgins eds. interactions directly as they happen. The technique is well (1984); Transcription And Translation, B. D. Hames & S. J. Suited to determination of kinetic parameters. Comparative Higgins eds. (1984); Culture Of Animal Cells, R.I. Freshney, affinity ranking is extremely simple to perform, and both Alan R. Liss, Inc., (1987); Immobilized Cells And Enzymes, kinetic and affinity constants can be derived from the sensor IRL Press, (1986); B. Perbal, A Practical Guide To Molecular gram data. 25 Cloning (1984); the treatise, Methods. In Enzymology, Aca When analyte is injected in a discrete pulse across a ligand demic Press, Inc., N.Y.; Gene Transfer Vectors For Mamma Surface, the resulting sensorgram can be divided into three lian Cells, J. H. Miller and M. P. Calos eds., Cold Spring essential phases: (i) Association of analyte with ligand during Harbor Laboratory (1987); Methods. In Enzymology, Vols. sample injection; (ii) Equilibrium or steady state during 154 and 155 (Wu et al. eds.): Immunochemical Methods In sample injection, where the rate of analyte binding is bal 30 Cell And Molecular Biology, Mayer and Walker, eds., Aca anced by dissociation from the complex; (iii) Dissociation of demic Press, London (1987); Handbook Of Experimental analyte from the surface during buffer flow. Immunology, Volumes I-IV. D. M. Weir and C. C. Blackwell, The association and dissociation phases provide informa eds., (1986); Manipulating the Mouse Embryo, Cold Spring tion on the kinetics of analyte-ligand interaction (k, and k, Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); the rates of complex formation and dissociation, k/k K). 35 and in Ausubel et al., Current Protocols in Molecular Biol The equilibriumphase provides information on the affinity of ogy, John Wiley and Sons, Baltimore, Md. (1989). the analyte-ligand interaction (K). General principles of antibody engineering are set forth in BIAevaluation software provides comprehensive facilities Antibody Engineering, 2nd edition, C. A. K. Borrebaeck, Ed., for curve fitting using both numerical integration and global Oxford Univ. Press (1995). General principles of protein fitting algorithms. With Suitable analysis of the data, separate 40 engineering are set forth in Protein Engineering, A Practical rate and affinity constants for interaction can be obtained Approach, Rickwood, D., et al., Eds. IRL Press at Oxford from simple BIAcore investigations. The range of affinities Univ. Press, Oxford, Eng. (1995). General principles of anti measurable by this technique is very broad ranging from mM bodies and antibody-hapten binding are set forthin: Nisonoff, to pM. A., Molecular Immunology, 2nd ed., Sinauer Associates, Sun Epitope specificity is an important characteristic of a 45 derland, MA (1984); and Steward, M. W., Antibodies, Their monoclonal antibody. Epitope mapping with BIAcore, in Structure and Function, Chapman and Hall, New York, N.Y. contrast to conventional techniques using radioimmunoassay, (1984). Additionally, standard methods in immunology ELISA or other surface adsorption methods, does not require known in the art and not specifically described are generally labeling or purified antibodies, and allows multi-site speci followed as in Current Protocols in Immunology, John ficity tests using a sequence of several monoclonal antibod 50 Wiley & Sons, New York; Stites et al. (eds), Basic and Clini ies. Additionally, large numbers of analyses can be processed cal-Immunology (8th ed.), Appleton & Lange, Norwalk, automatically. Conn. (1994) and Mishell and Shiigi (eds), Selected Methods Pair-wise binding experiments test the ability of two MAbs in Cellular Immunology, W.H. Freeman and Co., New York to bind simultaneously to the same antigen. MAbs directed (1980). against separate epitopes will bind independently, whereas 55 Standard reference works setting forth general principles MAbs directed against identical or closely related epitopes of immunology include Current Protocols in Immunology, will interfere with each other's binding. These binding John Wiley & Sons, New York: Klein, J., Immunology. The experiments with BIAcore are straightforward to carry out. Science of Self-Nonself Discrimination, John Wiley & Sons, For example, one can use a capture molecule to bind the New York (1982); Kennett, R., et al., eds., Monoclonal Anti first Mab, followed by addition of antigen and second MAb 60 bodies, Hybridoma: A New Dimension in Biological Analy sequentially. The sensorgrams will reveal: 1. how much of the ses, Plenum Press, New York (1980); Campbell, A., “Mono antigen binds to first Mab, 2. to what extent the second MAb clonal Antibody Technology” in Burden, R., et al., eds., binds to the surface-attached antigen, 3. if the second MAb Laboratory Techniques in Biochemistry and Molecular Biol does not bind, whether reversing the order of the pair-wise ogy, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunnol test alters the results. 65 ogy 4" ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Peptide inhibition is another technique used for epitope Barbara A. Osborne, H. Freemand & Co. (2000); Roitt, I., mapping. This method can complement pair-wise antibody Brostoff, J. and Male D. Immunology 6" ed. London: Mosby US 8,551,476 B2 103 104 (2001); Abbas A., Abul, A. and Lichtman, A., Cellular and was confirmed by immunohistochemistry studies of tissue Molecular Immunology Ed. 5, Elsevier Health Sciences Divi sections from the lateral ventricle region of P7 rat cortex. A sion (2005); Kontermann and Dubel, Antibody Engineering, majority of cortical cells that labeled with CC1 antibody also Springer Verlan (2001); Sambrook and Russell, Molecular labeled with anti-Sp35 antibody. Data not shown. The speci Cloning: A Laboratory Manual. Cold Spring Harbor Press ficity of the interaction was confirmed by preadsorption of the (2001); Lewin, Genes VIII, Prentice Hall (2003); Harlow and anti-Sp35 antibody with Sp35-Fc (see Example 2), which Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor eliminated the signal. Press (1988); Dieffenbach and Dveksler, PCR Primer Cold Sp35-specific RNAi Knockdown of Sp35 Expression Pro Spring Harbor Press (2003). motes Oligodendrocyte Growth and Differentiation All of the references cited above, as well as all references 10 Sp35-specific RNAi was used to ablate Sp35 expression in cited herein, are incorporated herein by reference in their oligodendrocyte precursor cells to examine how Sp35 con entireties. tributes to oligodendrocyte growth and differentiation. 50,000 A2B5 oligodendrocyte precursor cells were infected EXAMPLES with lentivirus carrying Sp35-specific RNAi sequence or con 15 trol RNAi prepared as follows. Example 1 Murine and rat Sp35 DNA sequences were compared to find homologous regions to use for candidate Small-hairpin Sp35 is Involved in Oligodendrocyte Biology RNAs (shRNA). CH324, for lentivirus expression of Sp35 RNAi, was constructed by annealing oligonucleotides LV1 Oligodendrocytes mature through several developmental 035 and LV1-036 and ligating to HpaI and XhoI digested stages from A2B5 progenitor cells (which express A2B5), pIL3.7. The plL3.7 vector, additional methodology and differentiating into pre-myelinating oligodendrocytes (which virus production were as described in Rubinson et al., Nat. express O1 and O4) and finally into mature myelinating oli Genet. 33,401-06 (2003). The Sp35 RNAi oligonucleotides godendrocytes (which express O1, O4 and MBP). Thus, by were purchased from MWG and have the following monitoring the presence and absence of the A2B5. O1, O4 25 sequences: LV1-035 (sense oligo) 5'-TGA TCG TCA TCC and MBP markers it is possible to determine a given cells TGCTAG ACT TCA AGAGAG. TCTAGC AGG ATGACG developmental stage and to evaluate the role of Sp35-Fc in ATC TTTTTT C-3' (SEQ ID NO:346) and LV1-036 (anti oligodendrocyte biology. For a general review of oligoden sense oligo) 5'-TCGAGAAAAAAG ATC GTC ATC CTG drocyte biology, see, e.g., Baumann and Pham-Dinh, Physiol. CTA GAC TCT CTTGAA GTC TAG CAG GAT GAC GAT Rev. 81:871-927 (2001). 30 CA-3' (SEQ ID NO:347). Monoclonal antibodies against O4, MBP and CNPase were Control RNAi was designed with the same oligonucleotide from Sternberger Monoclonals; antibody to APC (clone sequences except for the nucleotide changes indicated in CC-1; ref. 29) was from Calbiochem. Other antibodies were lower-case letters: 5'-TGA TCC TCATcCttC Tat ACT TCA to BIII tubulin (Covance), Sp35 (Biogen Idec), Fyn (Santa AGA GAG TgT AGC AGG ATG AcG ATC TTTTTT CTC Cruz, Biotechnology) and phospho-Fyn (Biosource). Mono 35 GA-3' (SEQID NO:348) and 5'-TCGAGAAAAAAG ATC clonal antibodies against A2B5 are available from Chemicon. GTC ATC CTG CTA GAC TCT CTTGAA GTa TAGaAG Sp35 is Expressed in Oligodendrocytes GAT GAC GAT CA-3'. (SEQID NO:349). The expression of Sp35 in purified rat P13 CG neuron, P2 Prior to producing the lentivirus, DNA from pl. L3.7 or oligodendrocyte, and P4 astrocyte cultures was analyzed by candidate shRNA in plL3.7 were cotransfected with murine polymerase chain reaction after reverse transcription (RT 40 Sp35-HA tagged plasmid at a ratio of 5 to 1 into CHO cells in PCR). A kit from Ambion, Inc. was used to extract mRNA a 6-well format. Knockdown was analyzed by western blot from the rat brain cells according to the manufacturers detection of Sp35-HA tag from transfected CHO cell lysates instructions. Semi-quantitative RT-PCR was carried out using as well as by northern blot of total RNA prepared from dupli forward primer 5' AGAGACATGCGATTGGTGA 3' (SEQ cate wells. The blot was probed with a fragment of Sp35 IDNO:344), and reverse primer 5' AGAGATGTAGACGAG 45 cDNA. Assays were performed 48 hours post-transfection. GTCATT 3' (SEQ ID NO:345) showed high expression in As expected, there was a 10-fold reduction of Sp35 mRNA in neurons, lower expression in oligodendrocytes, and no CH324 RNAi-treated CHO cells relative to control-treated expression in astrocytes. cells. Data not shown. RNAilentiviruses carrying green fluo The expression of Sp35 in oligodendrocytes was con rescent protein (GFP) were generated as described in Rubin firmed by in situ hybridization in sections derived from adult 50 son etal. In cultures treated with either control or Sp35 RNAi, rat optic nerve. Rat optic nerve sections were prepared and approximately 80% of the oligodendrocytes were GFP posi processed as described in Miet al., “Sp35 is a component of tive. Total cell number was not altered by the RNAi treat the Nogo-66 receptor/p75 signaling complex. Nat. Neurosci. ments. To quantify the effects of RNAi on differentiation, 7: 221-28 (2004) and probed with digoxigenin-labeled Sp35 only GFP-expressing oligodendrocytes were counted. antisense or sense RNAs using the first 500 nucleotides of the 55 Enriched populations of oligodendrocytes were grown Sp35 coding sequence. The sections were stained according from female Long Evans P2 rats as described by Conn, Meth. to the manufacturers instructions using a Tyramide Signal Neurosci. 2:1-4 (Academic Press: 1990) with modifications Amplification kit (Amersham BioSciences) and a fluorescent as follows. Briefly, the forebrain was dissected and placed in anti-digoxigenin conjugated antibody kit (PerkinElmer). For Hank’s buffered salt solution (HBSS: Invitrogen). The tissue combined in situ and immunofluorescence analyses, the sec 60 was cut into 1-mm fragments and was incubated at 37°C. for tions were first probed with digoxigenin-labeled RNAs and 15 min in 0.01% trypsin and 10 ug/ml DNase. Dissociated then with antibodies, e.g. CC1 antibody (Calbiochem; a cells were plated on poly-L-lysine-coated T75 tissue culture marker of mature oligodendrocytes) or anti-Sp35 antibody. flasks and were grown at 37°C. for 10 d in DMEM medium We observed that oligodendrocytes that hybridized to an anti with 20% fetal calf serum (Invitrogen). Oligodendrocyte pre sense Sp35 probe also co-stained with an antibody to CC1 65 cursors (A2B5) were collected by shaking the flask over (data not shown). No specific labeling was observed using a night at 200 rpm at 37°C., resulting in a 95% pure population. sense Sp35 probe. Sp35 expression in oligodendrocytes also Cultures were maintained in high-glucose Dulbecco's modi US 8,551,476 B2 105 106 fied Eagle's medium (DMEM) with FGF/PDGF (10 ng/ml; GTC GAC GCG GCC GCATGCTGG CGG GGG GCGT-3' Peprotech) for 1 week. Removal of FGF/PDGF allowed (SEQ ID NO:354) and reverse primer 5'-CAG CAG GTC A2B5 cells to differentiate into O4" premyelinating oligo GAC CTC GCC CGG CTG GTT GGC CAA CCA GCC dendrocytes after 3-7 d, and to differentiate into O4" and GGG CGAGGT CGA CCT CGA GG-3' (SEQID NO:355). MBP" mature oligodendrocytes after 7-10 d. These differen 5 The blunt-end PCR product was subcloned into the SrfI site tiation states are readily apparent from changes in morphol of the PCR SCRIPTAMP vector (Stratagene) to create PCR ogy: A2B5 cells are bipolar in shape, O4" premyelinating SCRIPTAMP-Sp35. A Sall fragment was isolated from PCR oligodendrocytes have longer and more branched processes SCRIPTAMP-Sp35 and subcloned into the PCRCAMP Ig and MBP mature oligodendrocytes contain myelin sheet vector (derivative of Stratagene vector PCR SCRIPTAMP). structures between processes. 10 In the PCRCAMP Ig vector, the hinge and Fc gamma A2B5 oligodendrocyte precursor cells were infected with sequence is subcloned as a SalI(5') to NotI(3') fragment. The the lentivirus containing the CH324 RNAi. The resulting cells SalI Sp35 fragment was subcloned into the SalI site of the were cultured for 3 days and the number of O4-positive (a PCRCAMP Ig vector thereby fusing the Sp35 signal marker for oligodendrocyte differentiation) oligodendrocytes sequence and extracellular domain (codons 1-532) in-frame was counted. Endogenous Sp35 expression was reduced by 15 with sequences encoding the hinge and Fc region of human infection with Sp35 RNAi lentivirus and was confirmed by Ig1. Correct isolates were identified, and a NotI fragment RT-PCR. Reduction of Sp35 resulted in more highly differ encompassing the Sp35 Fc fragment was subcloned into the entiated, mature oligodendrocytes as compared with control single NotI cloning site of the CHO expression vector, PV90 infected cells, as was evident by increases in the length of cell (Biogen Idec). The resulting plasmid was confirmed by DNA processes and by the presence of abundant myelin sheet struc sequencing and designated GT123. tures (data not shown). In cells that expressed Sp35 RNAi, Stable cell lines expressing the Sp35-Fc fusion protein there were three times as many mature (O4-positive) oligo were generated by electroporation of CHO host cells DG44 dendrocytes as in control cultures. These data indicate that with plasmid GT123. Transfected CHO cells were cultured in Sp35 may negatively regulate oligodendrocyte differentia alpha minus MEM in the presence of 10% dialyzed serum and tion. 25 4 mM glutamine to select for nucleoside-independent growth. Dominant-Negative Sp35 Promotes Oligodendrocyte Fourteen days post-transfection, cells were fed fresh media. Growth and Differentiation To screen for cells expressing Sp35-Fc. CHO cells were Lentiviral vectors that express wild-type and a dominant labeled with phycoerythrin (PE)-labeled goat anti-human negative form of Sp35 were constructed. DNA sequence IgG (Jackson Labs) and Subjected to high speed flow cytom encoding mouse full length Sp35 (FL-Sp35, amino acid resi 30 etry sorting in a FACS Mo-Flo (Cytomation). The cells that dues 34-614 of SEQID NO:2) was amplified by PCR using expressed the highest levels of Sp35-Fc were selected. These primers 5'-GAG GATCTCGACGCG GCC GCATGGAGA cells were expanded in culture for 7 days, then re-labeled and CAG ACA CACTCCTG-3' (SEQID NO:350) and 5'-GGG re-sorted. Cells expressing the highest levels of Sp35-Fc were GCG GAATTG GAT CCT CACAGATCCTCTTCT GAG isolated as individual clones in 96-well plates. These clones ATGAG-3' (SEQ ID NO:351) and inserted into the HRST 35 were grown for two weeks and then fed fresh media one day IRESeGFP lentiviral vector at the Not and BamHI sites. prior to FACS analysis to check for expression levels. Clones Similarly, DNA sequence encoding dominant negative Sp35 that expressed the highest levels of Sp35-Fc were expanded, (DN-Sp35, amino acid residues 34-581 of SEQID NO:2) was and frozen cell banks were established. The cell lines were amplified by PCT using primers 5'-GAG GAT CTC GAC adapted to grow in Suspension culture in the serum-free media GCG GCC GCA TGG AGA CAG ACA CAC TCC TG-3' 40 BCM16. The titer of Sp35-Fc produced by these clones was (SEQ ID NO:352) and 5'-GAT ACG GAT CCT CAGCCT determined by growing cell lines at 37°C. for 4-5 passages, TTG CCC CGG CTC CAT AGA AAC AGC-3' (SEQ ID then growing the cells to 50% maximal cell density and NO:353). The FL-Sp35 and DN-Sp35 plasmids were trans culturing them for 10-15 days at 28°C. until the viable cell fected into 293 cells to produce lentivirus as described by density dropped to 75%. At this time, the culture media were Rubinson et al., “A lentivirus-based system to functionally 45 harvested, cleared of cells and debris by centrifugation, and silence genes in primary mammalian cells, stem cells and the culture supernatants titered for Sp35-Fc levels by Western transgenic mice by RNA interference.” Nat. Genet. 33: 401 blot analysis using an anti-human Ig antibody (Jackson Lab) 06 (2003). Oligodendrocytes were infected with lentivirus at as the probe. 2 MOI per cell and confirmed expression of FL-Sp35 and Sp35-Fc fusion protein was purified from the clarified cul DN-Sp35 by western blot. 50 ture medium as follows: 9 ml of 1M HEPES pH 7.5 was added DN-Sp35 promoted oligodendrocyte differentiation, pro to 900 ml of conditioned medium. The medium was batch ducing an increase in the number of mature oligodendrocytes. loaded for 3 hr at 4°C. onto 3 ml of Protein A Sepharose In contrast, overexpression of full-length Sp35 (FL-Sp35) (Amersham Bioscience). The resin was collected in a 1.5 cm had the opposite effect and inhibited differentiation, as was (I.D.) column, and washed four times with 3 ml PBS, two evident by a reduction in the number of mature oligodendro 55 times with 4 ml of PBS containing 800 mM NaCl, and then cytes as compared with the control (data not shown). again with 3 ml of PBS. The Sp35-Fc was eluted from the column with 25 mMNaH2PO, pH 2.8 and 100 mM. NaCl in Example 2 1.5 ml fractions and neutralized by adding 75 ul of 0.5 M NaH2PO, pH 8.6. Peak protein-containing fractions were Construction and Purification of Sp35-Fc Fusion 60 identified by absorbance at 280 nm, pooled, and subjected to Protein further purification on a 1 mL Protein A column. Prior to loading, NaCl was added to 600 mM and HEPES, pH 7.5 to A construct was made fusing the extra-cellular portion of 50 mM. The column was washed twice with 600 ul of 10 mM human Sp35 (residues 1-532) to the hinge and Fc region of HEPES pH 7.5 and 1 M NaCl, and then with 1 ml PBS. human IgG1 to study the biological function of Sp35. A 65 Sp35-Fc was eluted from the column with 25 mMNaH2PO, partial coding sequence for human Sp35 was obtained by pH 2.8 and 100 mM NaCl, collecting 0.5 mL fractions, and PCR from clone 227.2 using the forward primer 5'-CAG CAG neutralized by adding 25ul of 0.5 MNaH2PO pH 8.6. Peak US 8,551,476 B2 107 108 protein-containing fractions were identified by absorbance at from the immunized mice were collected before the first 280 nm and pooled. By reducing SDS-PAGE, the Sp35-Fc immunization and 1 week after the second and third immu protein migrated as a single band (>95% pure) with an appar nizations, and anti-Sp35 antibody titers were measured by ent mass of 90 kDa. Under non-reducing conditions, the FACS assay on Sp35-expressing COS-7 cells as described protein ran as a dimer with an approximate mass of 180 kDa. above. A booster final dose was given after the third immu The purified Sp35-Fc protein was aliquoted and stored at nization and three days prior to when hybridoma fusions were -70° C. initiated. Sera from mice immunized with the various Sp35 peptides Example 3 were screened by ELISA as described above. Mice that were 10 positive for antibodies that specifically bound Sp35 express Production of Sp35-Specific Monoclonal Antibodies ing COS-7 cells were identified by flow cytometry (FACS) as Anti-Sp35 Antibodies that specifically bind an Sp35 described above, and were sacrificed. Splenocytes were iso polypeptide of the invention were made using the following lated from the mice and fused to the FL653 myeloma (an methods and procedures. 15 APRT-derivative of a Ig-/HGPRT-Balb/c mouse myeloma, A. Antibody Screening Assays maintained in DMEM containing 10% FBS, 4500 mg/L glu 1. ELISA Assay cose, 4 mM L-glutamine, and 20 mg/ml 8-azaguanine) as Sp35-Fc (0.5 ug in 50 ul of 0.1 M sodium bicarbonate described in Monoclonal Antibodies. Hybridomas: A New buffer, pH 9.0) was added to each well of 96-well Max Dimension in Biological Analyses, ed. Kennett, R. H. McK iSorpTM plates (NunctM). The plates were then incubated at earn, T.J. and Bechtol, K. B. New York: Plenum Press (1982). 37°C. for 1 hour or 4°C. for 16 hours. Non-specific binding Fused cells were plated into 24- or 48-well plates (Corning sites on the plates were blocked using 25 mM HEPES, pH 7.4 Glass Works, Corning, N.Y.), and fed with adenine, aminop containing 0.1% BSA, 0.1% ovalbumin, 0.1% (5% (w/v) terin and thymidine (AAT, available from Sigma(R) Chemical nonfat dry milk in 150 mM NACE) and 0.001% azide. Dilu Co., St. Louis, Mo.) containing culture medium. AAT resis tions of serum or hybridoma Supernatants (for example, serial 25 tant cultures were screened by ELISA or flow cytometry as three-fold dilutions) were added across each row of the plate, described above for binding to either Sp35-COS-7 cells or to and incubated at 25°C. for 1 hour. After washing three times Sp35-Fc. Positive hybridomas were further subcloned by lim with PBS, 50 ul of a 1:10,000 dilution of horseradish peroxi iting dilution. dase-conjugated goat anti-mouse secondary antibody (Jack Seventeen hybridoma cell lines producing monoclonal son ImmunoResearch Inc.) was added to each well and incu 30 antibodies produced from mice immunized with Sp35-Fc bated further for 1 hour. After three washings, color was were isolated. Properties of the hybridoma-derived mono developed by TMB (Pierce) and stopped with 2 M sulfuric clonal antibodies are shown in Tables 3A and 3B. acid. Colorintensity was monitored in a spectrophotometer at Polynucleotides encoding the variable domains (V, and 450 nm. V.) of monoclonal antibodies 1A7, 2F3, 3P1D10.2C3 and 2. FACS Assay 35 3P1E1 1.3B7 were isolated by PCR, cloned and were sub COS-7 cells were labeled with 0.1 uM CellTrackerTM jected to sequence analysis by the following method. Total Green CMFDA (Molecular Probes, Eugene, Oreg.) as RNA was extracted from hybridoma cells using QiagenR) described by the vendor. Equal volumes of CellTrackerTM RNeasy(R) minikit and cDNA was generated from the isolated labeled control cells were mixed with washed Sp35-COS-7 RNA by RT PCR, using standard conditions. A cocktail of cells (produced by transient transfection of Sp35 expression 40 primers were used for the RT-PCR. A preferred set of primers vector) before incubation with anti-Sp35 test sera or hybri included a primer with the 5' of the primer hybridizing to the doma supernatants. Fifty microliters of the cell mixture was signal sequence and the 3' end of the primer hybridizing to the dispensed into each well of a 96-well V-bottom polystyrene constant domain 3' of the FR4/constant domainjunction. This plates (Costar R 3877, Corning, N.Y.) and 100 ul of mouse allows for the amplification of an intact variable domain with serum, hybridoma Supernatant, or a control anti-Sp35 anti 45 no ambiguities about the monoclonal antibody N-terminus body was added. After incubation at 4°C. for 30 minutes, the and the V/C junction. One of skill in the art will recognize that cells were washed and incubated with 50 ul of phycoerythrin primer sets need to be modified for amplifying different tem conjugated affinity pure F(ab')2 fragment goat anti-mouse plates and for different PCR conditions. Occasionally, the IgG Fc gamma specific second antibody (1:200, Jackson presence of highly abundant nonproductive messages (e.g. ImmunoResearch Laboratory, West Grove, Pa.) in PBS. At 50 the CDR3-FR4 frameshifted nonproductive light chain from the end of the incubation, the cells were washed twice with the fusion partner) or nonspecific productive messages can be PBS and suspended in 200 ul of PBS containing 1% fetal produced and complicate the cloning of variable chains. One bovine serum (FBS), and subjected to FACS analyses. Alter Solution is to use N-terminal sequence data from the authentic nately, Sp35-COS-7 cells were mixed with mouse serum or purified antibody to design a degenerate primer to enable hybridoma Supernatant and then treated with R-phycoeryth 55 cloning. Alternatively, one can use “universal framework rin-conjugated goat anti-mouse secondary antibody and primers, such as those described in Orlandi et al., PNAS directly subjected to standard FACS analyses. 86:3833 (1989), which “fix” the N- and C-termini of the B. Hybridoma Production of Murine Monoclonal Anti-Sp35 variable domains (i.e. the N-terminus of FR1 and the C-ter Antibodies minus of FR4 are primer-determined). Eight-week-old female RBF mice (Jackson Labs, Bar Har 60 Additionally, sequence data, for designing more effective bor, Me.) were immunized intraperitoneally with emulsion primers, can be obtained from the bulk RT-PCR products containing 50 ug Sp35-Fc (amino acids 34 to 532 of SEQID which have been gel purified and then sequenced. The PCR NO:2 fused to the hinge and Fc region of human IgG1), product can also be subcloned using, for example, the TOPO produced as described in Example 2 or were immunized Cloning Kit (Invitrogen) then sequence. Sequence data is intraperitoneally with an emulsion containing 50 ug of human 65 then obtained from multiple independent subclones or gel Sp35-Fc, and 50 ul complete Freund's adjuvant (Sigma(R) purified fragments to firmly establish the consensus Chemical Co., St. Louis, Mo.) once every two weeks. Sera Sequence. US 8,551,476 B2 109 110 The sequence of the light chain of the P1E1 1.3B7 was respectively) were co-transfected into 293-EBNA cells. determined by using a cocktail of 5' murine kappa light chain Western blot analysis (probed with human IgG-specific signal sequence primers: (i) 5' GGG GAT ATC CAC CAT reagents) of conditioned medium from transiently transfected GGA TTT TCA GGT GCA GAT TTT CAG 3' (SEQ ID cells confirmed the expression of chimeric 3P1E1 1.3B7 NO:356), (ii) 5' GGG GAT ATC CAC CAT GRA GTCACA huIgG1, kappa mAb. The resulting 3P1E1 1.3B7VH and VL KACYCA GGT CTTYRTA 3 (SEQ ID NO:357), (iii) 5’ polypeptide sequences are shown in Tables 6 and 8 and are GGG GATATC CAC CAT GAA GTT GCCTGT TAG GCT SEQID NOs: 173 and 209, respectively. The heavy and light GTTG 3' (SEQID NO:358), and (iv) 5' GGG GAT ATC CAC chain sequences for the 1A7, 2F3, and 3P1D10.2C3 mono CATGAGGKCCCCWGCTCAGYTYCT KGGA3' (SEQ clonal antibodies were determined by similar methods. ID NO:359), with a single 3' murine kappa constant domain 10 C. Identification of Anti-Sp35 Monoclonal Antibodies by primer: 5' GCGTCT AGA ACT GGA TGGTGG GAG ATG Phage Display GA 3' (SEQ ID NO:4), where K=G/T, R=A/G, W=A/T and Y-C/T. The resulting PCR product was subcloned and mul Anti-Sp35 monoclonal antibody Fab fragments were iden tiple independent Subclones were sequenced. The deduced tified and isolated from phage display libraries as described in consensus sequence was consistent with the Edman degrada 15 Hoet et al., Nat. Biotech. 23:344-348 (2005); Rauchenberger, tion sequencing data. Sequencing indicated that the degener et al., J. Biol. Chem. 278:194-205 (2003); and Knappik, et al., ate signal sequence 5' primer 5' GGG GAT ATC CAC CAT J. Mol. Biol. 296:57-86 (2000), all of which are incorporated GRA GTCACA KACYCA GGT CTTYRT A3' (SEQ ID herein by reference in their entireties. NO:357) was the one that had yielded the 3P1E1 1.3B7 light The MorphoSys Fab-phage display library HuCAL(R) chain variable domain during the amplification. GOLD (“Phage Display Library-2 in Table3B), which com The 3P1E1 1.3B7 heavy chain sequence was determined prises humanized synthetic antibody variable regions was using a cocktail of murine heavy chain signal sequence 5' screened against recombinant human soluble Sp35-Fc pro PCR primers: (i) 5' GGG GAT ATC CAC CAT GGRATG tein by standard ELISA AND IHC screening methods. See, SAG CTG KGT MATSCT CTT3', (SEQID NO:360) (ii) 5' e.g., Ostendorp, R., Frisch, C. and Urban M. “Generation, GGG GATATC CAC CAT GRACTTCGGGYT GAG CTK 25 engineering and production of human antibodies using GGTTTT3' (SEQID NO:361), and (iii) 5’ GGG GAT ATC HuCAL(R). Antibodies, Volume 2 Novel Technologies and CAC CATGGCTGT CTTGGGGCT GCTCTTCT3' (SEQ Therapeutic Use. New York: Kluwer Academic/Plenum ID NO:362), with a degenerate murine IgG CH1 constant 13-52 (2004). Fab-phages that specifically bound to Sp35 domain 3' primer 5' AGG TCT AGA AYC TCC ACA CAC were purified and characterized. Properties of these phage AGGRRCCAGTGGATAGAC3' (SEQID NO:363), where 30 display-derived monoclonal antibody Fab fragments are K=G/T, M=A/C, R=A/G, andY=C/T. PCR using this cocktail of primers, with a variety of different cycling conditions, shown in Table 3B as “phage display library-2-derived mono failed to yield a heavy chain variable domain sequence in clonal Fab fragments. Isolated Fab-phage 1968 was selected which the deduced N-terminus was consistent with that deter for further analysis. mined by Edman degradation sequence of the purified 35 Example 4 3P1E1 1.3B7 antibody. Wetherefore used heavy chain univer sal primers: FR15'AGGTSMARCTGCAGSAGT CWG G 3' (SEQID NO:364) and FR45' TGAGGA GACGGT GAC Immunoprecipitation of Sp35 by Anti-Sp35 CGT GGT CCCTTG GCC CCA G 3' (SEQ ID NO:365), Monoclonal Antibodies where M=A/C, R=A/G, S=C/G, and W=A/T. This set yielded 40 a murine heavy chain variable domain whose deduced To perform the immunoprecipitation, COS-1 cells express sequence was consistent with the empirical 3P1E1 1.3B7 ing Sp35, fused to a hemaglutinin (HA) tag on the N-termi data. nus, were produced by transiently transfecting COS-1 cells In order to verify that the heavy chain variable domain N with a DNA construct which expresses the full-length Sp35 and C-termini were authentic and not primer-determined, 45 protein with an HA tag. Cells were harvested 48 hr after another PCR reaction was performed with a degenerate signal transfection and were lysed in 1 ml lysis buffer (50 mM sequence primer 5' ATG GARTGY AAY TGG ATH CTN HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl, 1 mM CCN TTY A3' (SEQ ID NO:366) and the aforementioned EGTA, 1% Triton X-100 and 10% glycerol) for 30 min at 4° constant domain 3' primer 5'AGGTCT AGAAYCTCCACA C. After centrifugation at 14,000xg for 15 min, the superna CACAGGRRCCAGTGGATAGAC 3' (SEQID NO:367), 50 tants were incubated with Protein A/G-Sepharose beads where H=A/C/T, N=A/C/G/T, R=A/G, and Y=C/T. The (Santa Cruz) at 4°C. for 1 hr., and then incubated at 4°C. for design of the degenerate signal sequence primer was based 1 hr with either the 1A7 or the 2F3 anti-Sp35 murine mono upon signal sequences of the best hits derived from a TFASTA clonal antibodies. The beads were washed 3 times with lysis search of the Genbank rodent sequence database queried with buffer, boiled in Laemmlisample buffer, subjected to 4-20% the 3P1E1 1.3B7 consensus deduced FR1 sequence from the 55 PCR reaction with the “universal primer described above. SDS-PAGE, and analyzed by Western blotting using an anti This PCR yielded a product with a complete murine heavy body which recognizes the HA tag. As shown on the SDS chain variable domain. PAGE gel, monoclonal antibodies 1A7 and 2F3, immunopre The complete 3P1E1 1.3B7 murine variable domains were cipitated human and murine Sp35 (FIG. 1). As shown in FIG. used (with silent mutagenesis as necessary to introduce 60 1, monoclonal antibody 2F3 strongly immunoprecipitated restriction sites) in conjunction with human IgG1 and kappa both human and murine Sp35, while monoclonal antibody constant domain cDNAS to construct chimeric heavy and 1A7, which strongly immunoprecipitated human Sp35, only light chain cDNAs, respectively. The full-length immunoglo recognized murine Sp35 protein weakly. Similarly, mono bulin cDNAs were subcloned into an expression vector called clonal antibodies 1G7, 2B10, 2F3, 3P4C2.2D2, 3P4C8.2G9, pNE001, a derivative of the commercial EBV mammalian 65 L100, Li03, Li05, Li06, Li07, Li08, Lil 1 and Li12 immuno cell episomal expression vector pCEP4. The heavy and light precipitate human or mouse or human and mouse Sp35 (See chain expression vectors (called pXW372 and pXW363, Table 3B). Additionally, Li08 immunoprecipitates AP-Sp35 US 8,551,476 B2 111 112 and monoclonal antibodies 1B6.4 and 3E3. 1 immunoprecipi FBS/PBS, cells were fixed in 2% PFA and subjected to FACS tate endogenous Sp35 (See Table 3B). analysis by PE.) FACS result showed that MAbs 1A7 and 2F3 bound to COS-7 or 293 cells expressing Sp35, but not bind to Example 5 control cells with no Sp35 expression (FIG. 2). Anti-Sp35 Antibody Binding Specifically to Sp35 Example 7 Determined by ELISA Neurite Outgrowth Assay In order to determine which regions of the Sp35 polypep tide were bound by the various hybridoma- and phage dis 10 To test the ability of the hybridoma-derived and Fab-ph play-derived monoclonal antibodies produced in Example 2. age-derived monoclonal antibodies produced above to an ELISA assay was performed using a panel of truncated reverse the inhibitory effect of CNS myelin inhibitors, e.g., Sp35 polypeptides, each fused to the hinge and Fc regions of OMgp, on neurons, Lab-TekR) culture slides (4 wells) were IgG1 by the methods described in Example 1. The panel coated with 0.1 mg/ml poly-D-lysine (Sigma(R). Ap-OMgp consisted of the following Sp35 fragments: amino acids 15 (1 lug/spot) or PBS was spotted as 3 ul drops. Lab-TekR slides 34-425 of SEQ ID NO:2, amino acids 417-532 of SEQ ID were then rinsed and coated with 10 ug/ml laminin NO:2, amino acids 417-493 of SEQ ID NO:2, and amino (GibcoTM). Dorsal root ganglions (DRG's) from P6-7 Spra acids 34-532 of SEQ ID NO:2. Ovalbumin and BSA were gue Dawley rat pups were dissociated with 1 mg/ml collage used as controls. As shown in Table 3B. hybridoma-derived nase type 1 (Worthington), triturated with fire-polished Pas mAbs 2F3, 2B10, 3A3, 3P4c2.2d2, and 3P4c8.2 g9, and teur pipettes pre-plated to enrich in neuronal cells and finally Fab-phage derived mAbs 3383, 3563, 3564, 3565, 3568, plated at 10,000 cells/well on the pre-coated Lab-TekR cul 3569,3570, and 3582 all specifically bound to the 1-417 and ture slides. Tenug/ml of mAb 1A7 or 2F3 were added imme 1-534 Sp35 fragments, suggesting that these antibodies bind diately after plating of the DRGs. The culture medium was to epitopes in the LRR region of Sp35. Hybridoma-derived F12 (available from Gibco/Invitrogen) containing 5% heat Mabs 1A7, 3P1B11F9, 3P1D10.2C3, 3P1E11.3B7, 25 inactivated donor horse serum, 5% heat inactivated fetal 3P2C63G10.2H7, 2P2C9.2G4, 3P4A61D9, and 394C51D8, bovine serum and 50 ng/ml mouse nerve growth factor and Fab-phage-derived Mabs 3495, 3566, 3567, and 1968 (mNGF) and incubated at 37° C. and 5% CO, for 6 hours. specifically bound to the 34-532 Sp35 fragment and weakly Following incubation, the slides were fixed in 4% paraform bound to the 417-532 Sp35, suggesting that these antibodies aldehyde/20% sucrose and stained with anti-fIII-tubulin likely bind to epitopes which at least include a portion of Sp35 30 TUJ1 antibody (Covance) after 16 hours. C-terminal to the LRR region. In similar experiments, these As secondary antibody anti-mouse Alexa-Fluorr. 594 latter antibodies also specifically bound an Sp35 polypeptide (Molecular Probes) diluted 1:300 was added to the slides and consisting of amino acids 34-534 of human Sp35 and low incubated for 2 hours at room temperature. The slides were affinity to mouse and rat Sp35. The affinity of these latter coverslipped with Gel/MountTM (BiomedaTM). 5x digital antibodies for mouse and rat Sp35 was restored to the level 35 images were acquired with OpenLabTM software (Improvi seen using human Sp35 when amino acid 419 of the mouse or Sion, Inc., Lexington, Mass.), and the images were analyzed rat Sp35 is changed from histidine (H) to arginine (R). Argi for quantification of neurite outgrowth using the OPEN nine is the amino acid at position 419 inhuman Sp35. The K, LABTM software, all according to manufacturer's specified for monoclonal antibody 1A7 was determined to be 10 nM parameters. (1x10M) for binding human Sp35 and 20 uM (2x10 M) 40 Both MAbs 1A7 and 2F3 protected DRG neurons from for binding murine Sp35. For Ap-Sp35 ELISA to detect the OMgp-mediated inhibition of neurite outgrowth. (FIG. 3). antibodies bound to the 417 to 532 region, the ELISA was performed as follows: The Mabs were coated onto ELISA Example 8 plates, then incubated either with an Sp35-AP fusion protein at 4°C. overnight followed by AP-linked anti-human (H+L) 45 Monoclonal Antibody 1A7 Promotes Functional (1:5,000, Jackson ImmunoResearch) at RT for 1 hr., or with Recovery in the Rat Spinal Cord Injury Model AP-fusion proteins at 4°C. overnight. AP substrate was then developed by 10 mg/ml 4NPP in 0.1 M Glycin, 1 mM MgCl, Spinal cord injury (“SCI) was induced by dorsal over 1 mM ZnCl2 pH 10.5, and read at O.D. 405. hemi-section as follows, modified from methods described 50 previously (Li, S. etal. J. Neurosci. 24, 10511-10520 (2004)). Example 6 Anesthetized female Long Evans rats (7 weeks old, Charles River) were given pre-operative analgesia (Buprenorphine? Anti-Sp35 Antibody Binding Specifically to Sp35 Buprenex, 0.05 mg/kg, s.c.) and tranquilized (Midazolam, Determined by FACS 2.5 mg/kg i.p.) and a dorsal hemi-section was performed at 55 thoracic vertebra 6/7 completely interrupting the main dor To further characterize the binding properties of hybri somedial and the dorsolateral corticospinal tract (CST). The doma-derived anti-Sp35 mAbs 1A7 and 2F3 produced as dorsal and dorso-lateral components of the corticospinal tract described in Example 3, binding to both fixed and live COS-7 (CST) were completely interrupted and the ventral portion of or 293 cells expressing mouse or human Sp35 was compared. the CST left intact. The ventral tissue bridge remaining after Sp35 transfected and non-transfected cells were fixed and 60 hemi-section constituted approximately 20% of the cord in subject to FACS analysis (FACS: Cells transfected with both treatment groups (data not shown). human or mouse Sp35 or vector control were dissociated Hindlimb function was quantified using the Basso-Beattie from culture plates, washed with 2% FBS/PBS, and incu Bresnahan (BBB) open field scoring method (Eby, M. T. et bated with primary antibody at 1 lug/ml on ice for 1 hr. The al., J. Biol. Chem. 275, 15336-15342 (2000), incorporated cells were washed 3 times with 2% FBS/PBS, then incubated 65 herein by reference) and all animals Sustained marked func with PE labeled secondary antibody (1:100, Jackson Immu tional deficits after SCI, with almost complete hindlimb noResearch) on ice for 30 min. After 2 washes with 2% paralysis the day after surgery. Immediately after CST US 8,551,476 B2 113 114 transection, an intrathecal catheter was inserted into the Sub depending upon the antibody. One of skill in the art would be arachnoid space at T7 and connected to a primed mini-os able to determine an effective dose using assays described motic pump (Alzet model 2004, Alza Corp) inserted into the herein. Subcutaneous space. Mini-osmotic pumps delivered Human The culture medium was changed and the various mono IgG isotype control protein (5 mg/ml) or monoclonal anti clonal antibodies were replenished every three days. After 30 body 1A7 (4.8 mg/ml) continuously at a rate of 0.25ul/h over days at 37° C., the co-cultured cells were stained by immu 5 weeks. Control (Human IgG-treated) animals recovered nohistochemical staining (“IHC) for neurofilaments with substantial function over the 5 week duration of the experi anti-fII-tubulin antibody to identify axons, or anti-MBP ment, but plateaued at 3-4 weeks, ultimately attaining a mean antibody to identify oligodendrocytes (FIG. 4A-E). Co-cul 10 tured cells were also lysed and subjected to Western blot BBB score of 9-0.45 (FIG. 7). In contrast, continuous intrath analysis to quantify the MBP (FIG. 4G). Based on IHC and ecal infusion of 1A7 for 5 weeks after spinal cord transection Western blot analyses, co-cultured cells treated with anti resulted in significantly improved BBB scores over the con Sp35 antibodies 1A7 and 2F3 showed increased survival of trol animals by 5 weeks with a continued improvement in oligodendrocyte and neurons, increased numbers of bundled function in the 2-5 week timeframe, reaching a mean BBB 15 axons and increased numbers of MBP positive cells (FIG.4F, score of 11.1+0.7 (FIG. 4). These results demonstrate that 10-fold more MBP-positive cells when compared to control treatment with anti-Sp35 monoclonal antibody 1A7 pro antibody treated co-cultures. moted recovery of function after spinal cord injury as dem In a similar experiment, oligodendrocyte and DRG co onstrated by an increase in BBB score, axon regeneration and cultures were incubated in the presence or absence of anti less axon retraction observed by immunohistochemical stain Sp35 antibodies LiO5 and LiO6, or a negative control anti ing of the axons. body. Co-cultured cells were lysed and subjected to Western blot analysis to quantify the MBP (FIG. 8). Based on Western Example 9 blot analyses, co-cultured cells treated with anti-Sp35 anti bodies Li05 and LiO6 showed increased numbers of MBP Anti-Sp35 Antibodies 1A7, 2F3, 3P1D10.2C3, 25 positive cells, similar to co-cultured cells treated with 3, 10 3P1E1 1.3B7, 6P4F4.1D3, 6P4F4.1F9, 7P1D5.1G9, and 30 ug of Sp35-Fc (LINGO-1-Fc). LiO5, Li06, Li08, Li 13, Li28, Li33, D05 and D08 In similar experiments oligodendrocyte and DRG co-cul Promote Myelination. In Vitro tures were incubated in the presence or absence of anti-Sp35 antibodies 3P1D10.2C3, 3P1E1 1.3B7, 6P4F4.1D3, The role of anti-Sp35 antibodies 1A7 and 2F3 in myelina 30 6P4F4.1F9, 7P1 D5.1 G9, Li08, Li13, Li28, and Li33 and also tion was investigated in vitro by treating co-cultures of dorsal promoted myelination. Similarly, full-length antibodies D05 root ganglion (DRG) neurons and oligodendrocytes with and D08 also promoted myelination. anti-Sp35 antibodies 1A7 and 2F3 and testing for myelination These results indicated that treatment of DRG-oligoden by immunohistochemistry and Western blotting. For these drocyte cocultures with anti-Sp35 antibodies 1A7, 2F3, studies, it was necessary to first generate primary cultures of 35 3P1D10.2C3, 3P1E1 1.3B7, 6P4F4.1D3, 6P4F4.1F9, DRG neurons and of oligodendrocytes. 7P1 D5.1 G9, LiO5, LiO6, Li08, Li 13, Li28, Li33, D05 and Female Long Evans rat E14-E 17 embryonic dorsal root D08 promoted mature oligodendrocyte axon interactions and ganglia were cultured as described by Plant et al., J. Neurosci. myelination compared to control-antibody treated co-cul 22:6083-91 (2002). Dissected DRGs were plated on poly-L- tures. lysine-coated cover slips (100 ug/ml) for 2 weeks. The cells 40 were incubated in the presence of fluorodeoxyuridine for Example 10 days 2-6 and in NLA medium containing 1xE27, 100 ng/ml NGF (Gibco) for days 8-11. Anti-Sp35 Antibody 1A7 Promotes Oligodendrocyte Female Long Evans post-natal day 2 (P2) rat oligodendro Survival and Myelination. In Vivo cytes were cultured as described by Conn, Meth. Neurosci. 45 2:1-4 (Academic Press; 1990) with modifications as follows. Adult wild-type C57BL/6 male mice were fed cuprizone Briefly, the forebrain was extirpated from P2 rats and placed (0.2% milled with ground mouse chow by weight) for 6 in cold HBSS medium (Gibco). The tissue fragments were cut weeks to induce demyelination within the corpus callosum into 1 mm pieces and incubated at 37°C. for 15 min in 0.01% according to the method described by Morell Petal. Mol Cell trypsin and 10 ug/ml DNase. Dissociated cells were plated on 50 Neurosci. 12:220-7 (1998). Briefly, anti-Sp35 monoclonal a poly-L-lysine coated T75 tissue culture flasks and grown in antibody 1A7 was stereotactically injected into the demyeli DMEM with 20% fetal bovine serum at 37° C. for 10 days. nating corpus callosum at weeks 2, 2.5, and 3 weeks of A2B5-positive oligodendrocytes were collected by shaking cuprizone feeding, by the method described below. Control the flasks overnight at 200 rpm at 37°C. The A2B5 oligoden mice were stereotactically injected at the same intervals with drocytes were cultured for 7 days in DMEM (Gibco) contain 55 sterilized media containing control antibody. After the 6 ing 25 mM D-glucose, 4 mM L-glutamine, 1 mM sodium weeks of cuprizone feeding was completed, the mice were pyruvate, 50 lug/ml human apo-transferrin, 5 g/ml bovine returned to a normal diet for 2, 4 and 6 weeks (ground mouse pancreatic insulin, 30 nM sodium selenate, 10 nM hydrocor chow only) to allow remyelination. tisone, 10 nM D-biotin, 1 mg/ml BSA, 10 ng/ml FGF and The 1A7 and control monoclonal antibodies were deliv PDGF (Peprotech). The cells were then harvested by 60 ered as follows. The cuprizone-treated mice were anesthe trypsinization. The cells then co-cultured with the DRG neu tized with ketamine (80 mg/kg body weight) and xylazine (10 rons in the presence or absence of 1, 3, 10, or 30 ug/ml of anti mg/kg body weight) and positioned in an immobilization Sp35 monoclonal antibodies 1A7 or 2F3, or a negative con apparatus designed for stereotactic Surgery (David Kopf trol antibody in NLA medium containing 2% fetal bovine Instruments). The scalp was opened and the sterile com serum, 50 lug/ml ascorbic acid, 100 ng/ml NGF (Gibco). An 65 pounds injected (1 uM in 1 ml of HBSS) unilaterally into the effective antibody dose to administer in Such an assay has acutely demyelinated corpus callosum of the wild-type been determined to be in the range of 0.1 g/ml to 10 g/ml. recipient mice with a 10 ul Hamilton Syringe using stereotac US 8,551,476 B2 115 116 tic coordinates of 0.7 mm posterior and 0.3 mm lateral to compared to control-antibody or PBS treated animals, which bregma at a depth of 1.7 mm (Messier et al., Pharmacol. each only showed approximately 50% neuronal survival Biochem. Behav: 63: 313-18 (1999)). Additional control (FIG. 6). recipient mice were stereotactically injected with HBSS con taining no compounds. The opening in the skull was filled 5 Example 12 with Gelfoam, and the area was swabbed with penicillin and streptomycin (Gibco) and the wound was sutured. Mice were Testing Anti-Sp35 Antibodies for Remyelination in sacrificed every week of the experiment after injection and the Optic Nerve Crush Model their brains removed and processed for molecular, biochemi cal and histological analysis. 10 The right optic nerve receives complete crush by #5 for The animals receiving anti-Sp35 antibody 1A7 treatment ceps for 10 seconds around 1.5 mm behind the eyeball intraor showed increased mature oligodendrocyte Survival (based on bitally just before administration of 2 ul of monoclonal anti CC1 antibody staining, FIG. 5A) and axon myelination by body 1A7, 2F3, Li05 and LiO6 in 2 ml by intravitreal IHC using anti-MBP protein antibody or luxol fast blue (FIG. injection. 5B). CC1 antibody-positive oligodendrocytes were quanti 15 The animals receive a second intravitreal injection of the same treatment one week after the Surgery. Two weeks after tated at four weeks and 6 weeks (FIG. 5C). These results the surgery, the animals are perfused with EM fixatives, post indicated that anti-Sp35 antibody 1A7 treatment promoted fixed and processed for semithin and ultrathin sections. The mature oligodendrocyte Survival and axon myelination com longitudinal optic nerve sections are stained and prepared for pared to control-antibody treated mice. Similarly, animals 20 myelin observation. The myelination of the proximal and the receiving the 1A7 antibody in a lysolecithin model of demy distal parts of the crushed optic nerve are compared among elination also promoted axon myelination compared to con different treatment groups. Sp35-Fc and 1A7, 2F3, LiO5 and trol animals (data not shown). LiO6 treated animals, as well as appropriate controls, will be analyzed for remyelination in the distal part of the optic nerve Example 11 25 compared to the controls. Anti-Sp35 Antibody 1A7 Promotes Retinal Ganglion Example 13 Cell (RGC) Survival in the Optic Nerve Transection Model Testing Anti-Sp35 Antibodies for Axon Regeneration 30 in the Optic Nerve Crush Model Anti-Sp35 antibody 1A7 was tested in an optic nerve transection model, which investigates factors that affect neu The right optic nerve was crushed by #5 forceps for 10 ronal function. Young adult female Sprague Dawley (SD) rats seconds around 1.5-2 mm behind the eyeball intraorbitally were used in this study. The right optic nerve of each animal just before administration of 2 ug of monoclonal antibody was transected intraorbitally 1.5 mm from the optic disc. A 35 1A7 in PBS via intravitreal injection. 4 rats were tested with piece of gelfoam soaked with 6% Fluoro-Gold (FG) was the 1A7 antibody and 8 rats were used as control animals. The applied to the newly transected site right behind the optic disc animals received a second intravitreal injection of the same to label the surviving retinal ganglion cells (RGCs). The treatment one week after the Surgery. Three days prior to animals were divided into three groups (n-6 in each group) sacrifice of the test animals (day 11 of the experiment), 2 ml which received either anti-Sp35 antibody 1A7, control anti- 40 of CTB-FITC was injected intravitreally to label, antero body, or just PBS, by intravitreal injection. The volume of grade, the regenerative optic nerve axons. On the 14th day each intravitreal injection was 4 ul while the dosage of each post Surgery, the animals were perfused and postfixed. The injection was 2 ug. The intravitreal injections were performed crushed optic nerve was processed for frozen longitudinal immediately after the optic nerve transection. sections. The CTB-FITC labeled axons, which cross the All animals were allowed to survive for 1 week. Two days 45 lesion site were counted as regenerative fibers at various before sacrificing the animals, the left optic nerve of each distances beyond the crush site. When 1A7 was injected into animal was transected and 6% FG was administered as the eye, regeneration of axons was observed up to 250 um described above to label the surviving RGCs, to serve as the beyond the crush site. See FIG. 10. internal control. Animals were sacrificed with an overdose of Nembutal and the retinas dissected in 4% paraformaldehyde. 50 Example 14 Four radial cuts were made to divide the retinas into four quadrants (Superior, inferior, nasal and temporal). The retinas Anti-Sp35 Antibodies Promote Remyelination and were then post-fixed in the same fixative for 1 hour before Repair in the Optic Nerve Using the MOG Induced they were flat-mounted with the mounting medium (Dako). EAE Rat Model The slides were examined under a fluorescence microscope 55 using an ultra-violet filter (excitation wavelength=330-380 For theses experiments, the Myelin Oligodendrocyte Gly nm). Labeled RGCs were counted along the median line of coprotein (MOG) induced Experimental Autoimmune each quadrants starting from the optic disc to the peripheral Encephalomyelitis (EAE) rat model was used. This is the border of the retina at 500 um intervals, under an eyepiece animal model for human multiple sclerosis. 50 ul of 200 ng grid of 200x200 um. The percentage of surviving RGCs 60 complete Freund's adjuvant (Chondrex Inc.) plus 50 ul of 50 resulting from each treatment was expressed by comparing ug MOG in saline was emulsified (1:1) and kept on ice before the number of surviving RGCs in the injured eyes with their being injected intradermally at the base of the tail for each contra-lateral eyes. All data were expressed as mean-SEM. animal. Female brown Norway rats, 8-10 weeks old, were Statistical significance was evaluated by one way ANOVA, used for all experiments. General observation in the art indi followed by a Tukey-Kramer post hoc test. Differences were 65 cates that the EAE model is induced around 15 days after considered significant for p-0.05. Anti-Sp35 antibody 1A7 MOG injection. Rats are scored for clinical signs of EAE. The treated animals showed more neuronal survival (80%) when signs are scored as follows: grade 0.5, distal paresis of the tail; US 8,551,476 B2 117 118 grade 1, complete tail paralysis; grade 1.5, paresis of the tail cal signs; 1 limp tail: 2-hind limb weakness, impaired right and mild hind leg paresis; grade 2.0, unilateral severe hind leg ing reflex or waddled gait; 3-complete hind limb paralysis or paresis; grade 2.5, bilateral severe hind limb paresis; grade absent righting reflex: 4-complete hind limb paralysis with 3.0, complete bilateral hind limb paralysis; grade 3.5, com some degree of fore limb involvement; 5-animal fully para plete bilateral hind limb paralysis and paresis of one front lyzed: 6-moribund or dead) are recorded daily. All proce limb; grade complete paralysis (tetraplegia), moribund state, dures are performed following a protocol, approved by our or death. The animals receive treatment once the EAE model institutional animal care and use committee (IACUC). The is induced. animals receive the treatment with 1A7, 2F3, Li05 and LiO6 2 g/ul of an anti-Sp35 antibody (1 A7) was injected intra monoclonal antibodies or control antibody at day 0 of the vitreally at day 15 upon MOG-EAE induction. 2 g/ul of the 10 study. Blood samples are taken at various times throughout anti-Sp35 antibody, 1 A7, was injected two additional times at the experiments by retro-orbital bleeding technique. Plasma day 22 and day 28. Upon termination of the experiment, the is separated from PBMC by centrifugation and cell pheno animals were perfused with 4% PFA. The optic nerves were typing performed by FACS staining. Profiling of the humoral post fixed in 1% OsO4, dehydrated and embedded in Epon. anti-MOG antibody response is performed by ELISA using Semithin sections (1 uM) were cut and stained with Toluidine 15 Subclass-fisotype-specific mAbs (Pharmingen). At the end of blue forevaluation of myelination. The optic nerves of treated each experiment, brain, spinal cord, optic nerves and Sciatic animals were compared to untreated animals for axon regen nerves are harvested following perfusion. eration and remyelination in the optic nerve. All procedures This same protocol is used to induce the EAE in Sp35 were performed following a protocol approved by institu knockout mice and litter mates. Sp35 knockout mice typically tional animal care and use committee (IACUC). show lower EAE score (1.5), and no relapse compared to Animals receiving treatment with the anti-Sp35 antibody control (over a 45 day period), then wild type litter mates 1A7 showed remyelination and repair of the optic nerve as (EAE score 3.5). compared to normal optic nerves or animals which were Sp35-Fc and 1A7, 2F3 treated animals will be analyzed for subjected to MOG-induced EAE, but received no treatment remyelination comparing to the control. (FIG. 9). In FIG. 9C, the arrows point to myelinated axons. 25 The His-tagged MOGs protein was expressed in Pichia Animals receiving an antibody which recognizes domain III pastoris using a Doxycycline inducible TetO-AOX1 pro of Protein G from Streptococcus (MOPC21), not specific for moter (M. Levesque, D. Krushinskie and K. Strauch, manu Sp35, showed no signs of remylination or repair of the optic Script in preparation). The extracellular coding sequence nerve as compared to normal optic nerves or the optic nerves (Gly 1 through Gly125 of the mature protein after removal of of untreated animals (data not shown). The Sp35 antagonist 30 signal sequence) of rat MOG was PCR amplified using the antibody 1A7 promoted remyelination and repair of optic following primers: 5' GGGGTATCTCTCGAGAAAA nerves in a rat MOG-induced EAE optic neuritis model (FIG. GAGAGCATCATCATCATCATCATATGG 9). GACAGTTCAGAGT GATAGGG 3' (SEQID NO:368), and 5 TTCGCGGCCGCTATTAGCCAGGGTTG ATCCAGTA Example 15 35 GAAGGG3' (SEQID NO:369). The present invention is not to be limited in scope by the Testing Anti-Sp35 Antibodies for Promotion of CNS specific embodiments described which are intended as single Remyelination Using MOG Induced EAE Mouse illustrations of individual aspects of the invention, and any Model compositions or methods which are functionally equivalent 40 are within the scope of this invention. Indeed, various modi EAE is induced in the 129B6 mixed strain of mice by fications of the invention in addition to those shown and intradermal immunization (day 0) with 100 ug MOG1-125 described herein will become apparent to those skilled in the protein emulsified with complete Freund's adjuvant (CFA). art from the foregoing description and accompanying draw The injected volume is 100 ul per mouse and is distributed ings. Such modifications are intended to fall within the scope over 3 sites (pinnae, back and skin). The emulsion is prepared 45 of the appended claims. on the basis of a 1:1 Volume ratio and contains 1 mg/ml All publications and patent applications mentioned in this MOG1-125 and 2 mg/ml M. tuberculosis (strain H37Ra, specification are herein incorporated by reference to the same Chondrex). Pertussis toxin (200 ng/mouse) is administered extent as if each individual publication or patent application intra-peritoneally at the time of immunization and 2 days was specifically and individually indicated to be incorporated thereafter. Body weight and clinical EAE scores (0-no clini by reference.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 4 O9

<21 Os SEQ ID NO 1 &211s LENGTH: 1845 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1

atgctggcgg ggggcgtgag gagcatgcc C agcc.ccctcc tdgcctgctg gCagcc catc 60

CtcCtgctgg tectgggct C agtgctgtca ggct cqgc.ca cqggctgcCC go ccc.gctgc 12O

gagtgct cog cc caggaccg cgctgtgctg. tccaccgca agcgctttgt ggcagt cc cc 18O

US 8,551,476 B2 121 122 - Continued

Asn Glin Asp Glu Phe Ala Ser Phe Pro His Luell Glu Glu Luell Glu Luell 85 90 95

Asn Glu Asn Ile Wall Ser Wall Glu Pro Gly Ala Phe Asn Asn Luell 1OO 105 11 O

Phe Asn Luell Arg Thir Lell Luell Arg Ser ASn Arg Lell Luell Ile 115 12 O 125

Pro Luell Gly Wall Phe Thir Luell Ser Asn Luell Thir Luell Asp Ile 13 O 14 O

Ser Glu Asn Ile Wall Luell Luell Asp Tyr Met Phe Glin Asp Luell 145 150 155 160

Asn Luell Ser Lell Wall Gly Asp ASn Asp Lell Wall Tyr Ile 1.65 17O 17s

Ser His Arg Ala Phe Ser Luell Asn Ser Luell Glu Glin Luell Thir Luell 18O 185 19 O

Glu Cys Asn Lell Thir Ser Ile Pro Thir Glu Ala Lell Ser His Luell 195

His Gly Luell Ile Wall Lell Arg Luell Arg His Luell Asn Ile Asn Ala Ile 21 O 215 22O

Arg Asp Ser Phe Lys Arg Luell Tyr Arg Luell Wall Luell Glu Ile 225 23 O 235 24 O

Ser His Trp Pro Tyr Lell Asp Thir Met Thir Pro Asn Luell Tyr Gly 245 250 255

Lell Asn Luell Thir Ser Lell Ser Ile Thir His Asn Lell Thir Ala Wall 26 O 265 27 O

Pro Luell Ala Wall Arg His Luell Wall Luell Arg Phe Luell Asn Luell 285

Ser Tyr Asn Pro Ile Ser Thir Ile Glu Gly Ser Met Lell His Glu Luell 29 O 295 3 OO

Lell Arg Luell Glin Glu Ile Glin Luell Wall Gly Gly Glin Lell Ala Wall Wall 3. OS 310 315

Glu Pro Ala Phe Arg Gly Luell Asn Tyr Luell Arg Wall Luell Asn Wall 3.25 330 335

Ser Gly Asn Glin Lell Thir Thir Luell Glu Glu Ser Wall Phe His Ser Wall 34 O 345 35. O

Gly Asn Luell Glu Thir Lell Ile Luell Asp Ser ASn Pro Lell Ala Asp 355 360 365

Arg Luell Luell Trp Wall Phe Arg Arg Arg Trp Arg Lell Asn Phe Asn 37 O 375

Arg Glin Glin Pro Thir Cys Ala Thir Pro Glu Phe Wall Glin Gly Glu 385 390 395 4 OO

Phe Asp Phe Pro Asp Wall Luell Luell Pro ASn Tyr Phe Thir Cys Arg 4 OS 415

Arg Ala Arg Ile Arg Asp Arg Ala Glin Glin Wall Phe Wall Asp Glu 425 43 O

Gly His Thir Wall Glin Phe Wall Cys Arg Ala Asp Gly Asp Pro Pro Pro 435 44 O 445

Ala Ile Luell Trp Lell Ser Pro Arg His Luell Wall Ser Ala Ser 450 45.5 460

Asn Gly Arg Luell Thir Wall Phe Pro Gly Thir Lell Glu Wall Arg Tyr 465 470

Ala Glin Wall Glin Asp Asn Gly Thir Luell Cys Ile Ala Ala Asn Ala 485 490 495

Gly Gly Asn Asp Ser Met Pro Ala His Luell His Wall Arg Ser Ser SOO 505 51O US 8,551,476 B2 123 124 - Continued

Pro Asp Trp Pro His Gln Pro Asn Llys Thr Phe Ala Phe Ile Ser Asn 515 52O 525

Gln Pro Gly Glu Gly Glu Ala Asn Ser Thr Arg Ala Thir Wall Pro Phe 53 O 535 54 O

Pro Phe Asp Ile Llys Thr Lieu. Ile Ile Ala Thir Thir Met Gly Phe Ile 5.45 550 555 560

Ser Phe Leu Gly Val Val Lieu Phe Cys Lieu. Wall Lell Lell Phe Luell Trp 565 st O sts

Ser Arg Gly Lys Gly Asn. Thir Lys His Asn Ile Glu Ile Glu Tyr Wall 58O 585 59 O

Pro Arg Llys Ser Asp Ala Gly Ile Ser Ser Ala Asp Ala Pro Arg 595 6OO 605 Phe Asn Met Lys Met Ile 610

SEQ ID NO 3 LENGTH: 6 TYPE PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 3 Met Glin Val Ser Lys Arg 1.

SEQ ID NO 4 LENGTH: 29 TYPE: DNA ORGANISM: Murinae gen. sp.

< 4 OOs SEQUENCE: 4 gcgt.ctagaa citggatggtg ggagatgga 29

SEO ID NO 5 LENGTH: 15 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: WH-CDR1 (Li10)

SEQUENCE: 5 acttacccta tdgtt 15

SEQ ID NO 6 LENGTH: 5 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: WH-CDR1 (Li10)

SEQUENCE: 6 Thr Tyr Pro Met Val 1. 5

SEO ID NO 7 LENGTH: 51 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: WH-CDR2 (Li10)

SEQUENCE: 7 tggat.cggtc. Cttctggtgg C9ttactgct tatgctgact ccgittaaagg t 51 US 8,551,476 B2 125 126 - Continued

<210s, SEQ ID NO 8 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (Li10)

<4 OOs, SEQUENCE: 8 Trp Ile Gly Pro Ser Gly Gly Val Thr Ala Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 9 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (Li10)

<4 OOs, SEQUENCE: 9 C cct at agca gtggctggtg ggactitcgat Ctc 33

<210s, SEQ ID NO 10 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (Li10)

<4 OOs, SEQUENCE: 10 Pro Tyr Ser Ser Gly Trp Trp Asp Phe Asp Leu 1. 5 1O

<210s, SEQ ID NO 11 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO7)

<4 OOs, SEQUENCE: 11 atgtactitta tdggit 15

<210s, SEQ ID NO 12 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO7)

<4 OOs, SEQUENCE: 12 Met Tyr Phe Met Gly 1. 5

<210s, SEQ ID NO 13 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO7)

<4 OOs, SEQUENCE: 13 t citat citctic cittctggtgg citt tacttct tatgctgact c cqttaaagg t 51

<210s, SEQ ID NO 14 &211s LENGTH: 17 US 8,551,476 B2 127 128 - Continued

212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO7)

<4 OOs, SEQUENCE: 14 Ser Ile Ser Pro Ser Gly Gly Phe Thr Ser Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 15 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO7)

<4 OOs, SEQUENCE: 15 gatcgg catg cittittgatat c 21

<210s, SEQ ID NO 16 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO7)

<4 OOs, SEQUENCE: 16 Asp Arg His Ala Phe Asp Ile 1. 5

<210s, SEQ ID NO 17 &211s LENGTH: 14 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO5)

<4 OOs, SEQUENCE: 17 Cttacgct at gggit 14

<210s, SEQ ID NO 18 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO5)

<4 OOs, SEQUENCE: 18 Ala Tyr Ala Met Gly 1. 5

<210s, SEQ ID NO 19 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO5)

<4 OOs, SEQUENCE: 19 t citat cqttt cittctggtgg ctatactgat tatgctgact c cqttaaagg t 51

<210s, SEQ ID NO 2 O &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: US 8,551,476 B2 129 130 - Continued <223> OTHER INFORMATION: WH-CDR2 (LiO5)

<4 OOs, SEQUENCE: 2O Ser Ile Val Ser Ser Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 21 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO5)

<4 OOs, SEQUENCE: 21 gagggtgacc ataatgcttt tatatc 27

<210s, SEQ ID NO 22 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO5)

<4 OOs, SEQUENCE: 22 Glu Gly Asp His Asn Ala Phe Asp Ile 1. 5

<210s, SEQ ID NO 23 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (Li11)

<4 OOs, SEQUENCE: 23 t cittacgcta totat 15

<210s, SEQ ID NO 24 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (Li11)

<4 OOs, SEQUENCE: 24 Ser Tyr Ala Met Tyr 1. 5

<210s, SEQ ID NO 25 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (Li11)

<4 OOs, SEQUENCE: 25 t citat citcta cittctggtgg ctatactggit tatgctgact c cqttaaagg t 51

<210s, SEQ ID NO 26 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (Li11)

<4 OOs, SEQUENCE: 26 US 8,551,476 B2 131 132 - Continued

Ser Ile Ser Thr Ser Gly Gly Tyr Thr Gly Tyr Ala Asp Ser Val Lys 1. 5 1O Gly

<210s, SEQ ID NO 27 &211s LENGTH: 36 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (Li11)

<4 OOs, SEQUENCE: 27 gataccagcg ataatgacta citact acatg gacgt.c 36

<210s, SEQ ID NO 28 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (Li11)

<4 OOs, SEQUENCE: 28 Asp Thir Ser Asp Asn Asp Tyr Tyr Tyr Met Asp Wall 1. 5 1O

<210s, SEQ ID NO 29 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO1)

<4 OOs, SEQUENCE: 29 aagtaccaga tigact 15

<210s, SEQ ID NO 3 O &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO1)

<4 OOs, SEQUENCE: 30 Lys Tyr Gln Met Thr 1. 5

<210s, SEQ ID NO 31 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO1)

<4 OOs, SEQUENCE: 31 t citat citatic cittctggtgg caatactgtt tatgctgact c cqttaaagg t 51

<210s, SEQ ID NO 32 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO1)

<4 OOs, SEQUENCE: 32 Ser Ile Tyr Pro Ser Gly Gly Asn Thr Val Tyr Ala Asp Ser Val Lys 1. 5 1O US 8,551,476 B2 133 134 - Continued

Gly

<210s, SEQ ID NO 33 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO1)

<4 OOs, SEQUENCE: 33 gggact acag aggcagtictt tact ac 27

<210s, SEQ ID NO 34 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO1)

<4 OOs, SEQUENCE: 34 Gly. Thir Thr Glu Ala Val Phe Asp Tyr 1. 5

<210s, SEQ ID NO 35 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (Li12)

<4 OOs, SEQUENCE: 35 cagtacaata tottt 15

<210s, SEQ ID NO 36 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (Li12)

<4 OOs, SEQUENCE: 36 Gln Tyr Asn Met Phe 1. 5

<210s, SEQ ID NO 37 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (Li12)

<4 OO > SEQUENCE: 37 cgitat citctt cittctggtgg catgactato tatgctgact c cqttaaagg t 51

<210s, SEQ ID NO 38 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (Li12)

<4 OOs, SEQUENCE: 38 Arg Ile Ser Ser Ser Gly Gly Met Thr Met Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly US 8,551,476 B2 135 136 - Continued

<210s, SEQ ID NO 39 &211s LENGTH: 69 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (Li12)

<4 OOs, SEQUENCE: 39 gaag.cgttac ggcctt attg tagtggtggit agctgct act C cactact a citact acggit 6 O atggacgt.c 69

<210s, SEQ ID NO 4 O &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (Li12)

<4 OOs, SEQUENCE: 4 O Glu Ala Lieu. Arg Pro Tyr Cys Ser Gly Gly Ser Cys Tyr Ser Asp Tyr 1. 5 1O 15 Tyr Tyr Tyr Gly Met Asp Val 2O

<210s, SEQ ID NO 41 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO6)

<4 OOs, SEQUENCE: 41 gagtacccta tigat 15

<210s, SEQ ID NO 42 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO6)

<4 OOs, SEQUENCE: 42 Glu Tyr Pro Met Asp 1. 5

<210s, SEQ ID NO 43 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO6)

<4 OOs, SEQUENCE: 43 t citat c tatt cittctggtgg ctic tactgtt tatgctgact c cattaaagg t 51

<210s, SEQ ID NO 44 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO6)

<4 OOs, SEQUENCE: 44 Ser Ile Tyr Ser Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Ile Llys 1. 5 1O 15 US 8,551,476 B2 137 138 - Continued Gly

<210s, SEQ ID NO 45 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO6)

<4 OOs, SEQUENCE: 45 gagggtgact Ctgatgcttt tatatc 27

<210s, SEQ ID NO 46 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO6)

<4 OOs, SEQUENCE: 46 Glu Gly Asp Ser Asp Ala Phe Asp Ile 1. 5

<210s, SEQ ID NO 47 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO8)

<4 OOs, SEQUENCE: 47 Cattacgaga tiggitt 15

<210s, SEQ ID NO 48 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO8)

<4 OOs, SEQUENCE: 48 His Tyr Glu Met Val 1. 5

<210s, SEQ ID NO 49 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO8)

<4 OOs, SEQUENCE: 49 t citat cogitt cittctggtgg cqc tactaag tatgctgact c cqttaaagg t 51

<210s, SEQ ID NO 50 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO8)

<4 OOs, SEQUENCE: 50 Ser Ile Arg Ser Ser Gly Gly Ala Thir Lys Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly US 8,551,476 B2 139 140 - Continued <210s, SEQ ID NO 51 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-C DR3 (LiO8

<4 OOs, SEQUENCE: 51 gagt cqc.cag acgact actt tact ac 27

<210s, SEQ ID NO 52 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO8)

<4 OOs, SEQUENCE: 52 Glu Ser Pro Asp Asp Tyr Phe Asp Tyr 1. 5

<210s, SEQ ID NO 53 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO3)

<4 OOs, SEQUENCE: 53 Cagtacccta tigag 15

<210s, SEQ ID NO 54 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO3)

<4 OOs, SEQUENCE: 54 Gln Tyr Pro Met Glu 1. 5

<210s, SEQ ID NO 55 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO3)

<4 OO > SEQUENCE: 55 ggitatictato Cttctggtgg ctic tactgtt tatgctgact cogittaaagg t 51

<210s, SEQ ID NO 56 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO3)

<4 OOs, SEQUENCE: 56 Gly Ile Tyr Pro Ser Gly Gly Ser Thr Val Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 57 &211s LENGTH: 30 &212s. TYPE: DNA US 8,551,476 B2 141 142 - Continued <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO3)

<4 OO > SEQUENCE: 57 gcggggcagt gigctggggga Ctttgactac 3 O

<210s, SEQ ID NO 58 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO3)

<4 OOs, SEQUENCE: 58 Ala Gly Glin Trp Lieu. Gly Asp Phe Asp Tyr 1. 5 1O

<210s, SEQ ID NO 59 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO9)

<4 OO > SEQUENCE: 59 atgtacticta tdgtt 15

<210s, SEQ ID NO 60 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO9)

<4 OOs, SEQUENCE: 60 Met Tyr Ser Met Val 1. 5

<210s, SEQ ID NO 61 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO9)

<4 OOs, SEQUENCE: 61 tatat citctic cittctggtgg caagacitat tatgctgact c cqttaaagg t 51

<210s, SEQ ID NO 62 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO9)

<4 OOs, SEQUENCE: 62 Tyr Ile Ser Pro Ser Gly Gly Lys Thr Met Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 63 &211s LENGTH: 69 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO9) US 8,551,476 B2 143 144 - Continued

<4 OOs, SEQUENCE: 63 gatt cqagac gocggt atta cqatttittgg agtggittatc acaac tact a citact actac 6 O atggacgt.c 69

<210s, SEQ ID NO 64 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO9)

<4 OOs, SEQUENCE: 64 Asp Ser Arg Arg Arg Tyr Tyr Asp Phe Trp Ser Gly Tyr His Asn Tyr 1. 5 1O 15 Tyr Tyr Tyr Tyr Met Asp Val 2O

<210s, SEQ ID NO 65 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO4)

<4 OOs, SEQUENCE: 65 cgttacaata tiggit 15

<210s, SEQ ID NO 66 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO4)

<4 OOs, SEQUENCE: 66 Arg Tyr Asn Met Gly 1. 5

<210s, SEQ ID NO 67 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO4)

<4 OO > SEQUENCE: 67 gttatctato Cttctggtgg cqg tact cat tatgctgact cogittaaagg t 51

<210s, SEQ ID NO 68 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO4)

<4 OOs, SEQUENCE: 68 Val Ile Tyr Pro Ser Gly Gly Gly Thr His Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 69 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence US 8,551,476 B2 145 146 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO4)

<4 OOs, SEQUENCE: 69 tctatagoag atgatgcttt tdata 25

<210s, SEQ ID NO 70 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO4)

<4 OO > SEQUENCE: 7 O Ser Ile Ala Asp Asp Ala Phe Asp Ile 1. 5

<210s, SEQ ID NO 71 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO2)

<4 OOs, SEQUENCE: 71 acttacgaga tigatt 15

<210s, SEQ ID NO 72 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (LiO2)

<4 OOs, SEQUENCE: 72 Thr Tyr Glu Met Ile 1. 5

<210s, SEQ ID NO 73 &211s LENGTH: 48 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (Lif3)

<4 OO > SEQUENCE: 73 t citat cqgtc. cittctggtgg cct tacttgg tatgctgact c cqttaaa 48

<210s, SEQ ID NO 74 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR2 (LiO2)

<4 OOs, SEQUENCE: 74 Ser Ile Gly Pro Ser Gly Gly Lieu. Thir Trp Tyr Ala Asp Ser Val Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 75 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO2) US 8,551,476 B2 147 148 - Continued <4 OO > SEQUENCE: 75 atgt attact gtgtacggat tatgatagt agtggttggg Cttittgatat c 51

<210s, SEQ ID NO 76 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR3 (LiO2)

<4 OO > SEQUENCE: 76 Met Tyr Tyr Cys Val Arg Ile Asp Asp Ser Ser Gly Trp Ala Phe Asp 1. 5 1O 15

Ile

<210s, SEQ ID NO 77 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OO > SEQUENCE: 77 Asn Tyr Gly Met Asn 1. 5

<210s, SEQ ID NO 78 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OO > SEQUENCE: 78 Trp Ile Asn Thr Asp Thr Gly Glu Pro Thr Tyr Thr Glu Asp Phe Glin 1. 5 1O 15 Gly

<210s, SEQ ID NO 79 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OO > SEQUENCE: 79 Glu Gly Val His Phe Asp Tyr 1. 5

<210s, SEQ ID NO 8O &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OOs, SEQUENCE: 80 Phe Ser Asp Ala Trp Lieu. Asp 1. 5

<210s, SEQ ID NO 81 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OOs, SEQUENCE: 81 Glu Ile Arg Ser Lys Ala Asn. Asn His Ala Thr Asn Tyr Ala Glu Ser 1. 5 1O 15 Val Lys Gly

<210s, SEQ ID NO 82 US 8,551,476 B2 149 150 - Continued

&211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OOs, SEQUENCE: 82 Ser Phe Ala Tyr 1.

<210s, SEQ ID NO 83 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OOs, SEQUENCE: 83 Ser Ser Trp Thr Glin 1. 5

<210s, SEQ ID NO 84 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OOs, SEQUENCE: 84 Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Glin Llys Phe Lys 1. 5 1O 15 Gly

<210s, SEQ ID NO 85 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp. <4 OOs, SEQUENCE: 85 His Asn Ser Tyr Gly Met Asp Tyr 1. 5

<210s, SEQ ID NO 86 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WH-CDR1 (Li10)

<4 OOs, SEQUENCE: 86 cgggcgagtic aggg tattgg caactggitta gcc 33

<210s, SEQ ID NO 87 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (Li10)

<4 OO > SEQUENCE: 87 Arg Ala Ser Glin Gly Ile Gly Asn Trp Lieu Ala 1. 5 1O

<210s, SEQ ID NO 88 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (Li10)

<4 OOs, SEQUENCE: 88 gctgcatcca gtttggaaag t 21 US 8,551,476 B2 151 152 - Continued

<210s, SEQ ID NO 89 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (Li10)

<4 OOs, SEQUENCE: 89

Ala Ala Ser Ser Lieu. Glu Ser 1. 5

<210s, SEQ ID NO 90 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (Li10)

<4 OOs, SEQUENCE: 90 caac aggctic agactitt.ccc gct cacc 27

<210s, SEQ ID NO 91 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (Li10)

<4 OOs, SEQUENCE: 91

Glin Glin Ala Glin. Thir Phe Pro Lieu. Thir 1. 5

<210s, SEQ ID NO 92 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO7)

<4 OOs, SEQUENCE: 92 tctggagatc agttgggtga caaacatgtg gct 33

<210s, SEQ ID NO 93 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO7)

<4 OOs, SEQUENCE: 93 Ser Gly Asp Gln Lieu. Gly Asp Llys His Val Ala 1. 5 1O

<210s, SEQ ID NO 94 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO7)

<4 OOs, SEQUENCE: 94

Ctagacatta agaggc.ccgc a 21

<210s, SEQ ID NO 95 &211s LENGTH: 7 212. TYPE: PRT US 8,551,476 B2 153 154 - Continued <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO7)

<4 OO > SEQUENCE: 95 Lieu. Asp Ile Lys Arg Pro Ala 1. 5

<210s, SEQ ID NO 96 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO7)

<4 OOs, SEQUENCE: 96

Caggcgtggg acatcaagac ggtc 24

<210s, SEQ ID NO 97 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO7)

<4 OO > SEQUENCE: 97 Glin Ala Trp Asp Ile Llys Thr Val 1. 5

<210s, SEQ ID NO 98 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO5)

<4 OOs, SEQUENCE: 98 gggggagaca acattggaag talaga.gtgtc. cac 33

<210s, SEQ ID NO 99 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO5)

<4 OOs, SEQUENCE: 99 Gly Gly Asp Asn. Ile Gly Ser Lys Ser Val His 1. 5 1O

<210s, SEQ ID NO 100 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO5)

<4 OOs, SEQUENCE: 1.OO gatgattatg accggCCCtc a 21

<210s, SEQ ID NO 101 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO5)

<4 OOs, SEQUENCE: 101 US 8,551,476 B2 155 156 - Continued

Asp Asp Tyr Asp Arg Pro Ser 1. 5

<210s, SEQ ID NO 102 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO5)

<4 OOs, SEQUENCE: 102

Caggtgaggg acagcc.gtac taggaacgg gtg 33

<210s, SEQ ID NO 103 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO5)

<4 OOs, SEQUENCE: 103 Glin Val Arg Asp Ser Arg Thr Glu Glu Arg Val 1. 5 1O

<210s, SEQ ID NO 104 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (Li11)

<4 OOs, SEQUENCE: 104 cgggcgagtic aggagattgc caact actta gcc 33

<210s, SEQ ID NO 105 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (Li11)

<4 OOs, SEQUENCE: 105 Arg Ala Ser Glin Glu Ile Ala Asn Tyr Lieu Ala 1. 5 1O

<210s, SEQ ID NO 106 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (Li11)

<4 OOs, SEQUENCE: 106 gata cataca ctittgcagac t 21

<210s, SEQ ID NO 107 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (Li11)

<4 OOs, SEQUENCE: 107 Asp Thr Tyr Thr Lieu Gln Thr 1. 5 US 8,551,476 B2 157 158 - Continued <210s, SEQ ID NO 108 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (Li11)

<4 OOs, SEQUENCE: 108 caac aggctg acatttitc.cc gct ct ct 27

<210s, SEQ ID NO 109 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (Li11)

<4 OOs, SEQUENCE: 109 Glin Glin Ala Asp Ile Phe Pro Lieu. Ser 1. 5

<210s, SEQ ID NO 110 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO1)

<4 OOs, SEQUENCE: 110

Caggcgagtic aggacattag caact attta aat 33

<210s, SEQ ID NO 111 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO1)

<4 OOs, SEQUENCE: 111 Glin Ala Ser Glin Asp Ile Ser Asn Tyr Lieu. Asn 1. 5 1O

<210s, SEQ ID NO 112 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO1)

<4 OOs, SEQUENCE: 112 gatgcatcca atttggaaac a 21

<210s, SEQ ID NO 113 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO1)

<4 OOs, SEQUENCE: 113 Asp Ala Ser Asn Lieu. Glu Thr 1. 5

<210s, SEQ ID NO 114 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: US 8,551,476 B2 159 160 - Continued <223> OTHER INFORMATION: WL-CDR3 (LiO1)

<4 OOs, SEQUENCE: 114

Caac aggctg acaggttcCC toggtcact 3 O

<210s, SEQ ID NO 115 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO1)

<4 OOs, SEQUENCE: 115 Glin Glin Ala Asp Arg Phe Pro Ala Val Thr 1. 5 1O

<210s, SEQ ID NO 116 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO6)

<4 OOs, SEQUENCE: 116 cgggc.cagtic agagtattag tagctggttg gcc 33

<210s, SEQ ID NO 117 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO6)

<4 OOs, SEQUENCE: 117 Arg Ala Ser Glin Ser Ile Ser Ser Trp Lieu Ala 1. 5 1O

<210s, SEQ ID NO 118 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO6)

<4 OOs, SEQUENCE: 118 gctgcatcca gtttacgaac t 21

<210s, SEQ ID NO 119 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO6)

<4 OOs, SEQUENCE: 119 Ala Ala Ser Ser Lieu. Arg Thr 1. 5

<210s, SEQ ID NO 120 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO6)

<4 OOs, SEQUENCE: 120 ctacaagatt acagttaccc tict cact 27 US 8,551,476 B2 161 162 - Continued

<210s, SEQ ID NO 121 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO6)

<4 OOs, SEQUENCE: 121 Lieu. Glin Asp Tyr Ser Tyr Pro Leu. Thr 1. 5

<210s, SEQ ID NO 122 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO8)

<4 OOs, SEQUENCE: 122 caggcgagtic aggacattag titact attta aat 33

<210s, SEQ ID NO 123 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO8)

<4 OOs, SEQUENCE: 123 Gln Ala Ser Glin Asp Ile Ser Tyr Tyr Lieu. ASn 1. 5 1O

<210s, SEQ ID NO 124 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO8)

<4 OOs, SEQUENCE: 124 gatgitatic ca atttgcaaac a 21

<210s, SEQ ID NO 125 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO8)

<4 OOs, SEQUENCE: 125 Asp Wal Ser Asn Lieu. Glin Thr 1. 5

<210s, SEQ ID NO 126 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO8)

<4 OOs, SEQUENCE: 126 caac agtctgataatc tocc tict cact 27

<210s, SEQ ID NO 127 &211s LENGTH: 9 212. TYPE: PRT US 8,551,476 B2 163 164 - Continued <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO8)

<4 OOs, SEQUENCE: 127 Glin Glin Ser Asp Asn Lieu Pro Lieu. Thr 1. 5

<210s, SEQ ID NO 128 &211s LENGTH: 32 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO3)

<4 OOs, SEQUENCE: 128 gggcaagt ca gag cattagc agctatttaa at 32

<210s, SEQ ID NO 129 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO3)

<4 OOs, SEQUENCE: 129 Arg Ala Ser Glin Ser Ile Ser Ser Tyr Lieu. Asn 1. 5 1O

<210s, SEQ ID NO 130 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO3)

<4 OOs, SEQUENCE: 130 gctgcatcca gtttgcaaag t 21

<210s, SEQ ID NO 131 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO3)

<4 OOs, SEQUENCE: 131

Ala Ala Ser Ser Lieu. Glin Ser 1. 5

<210s, SEQ ID NO 132 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO3)

<4 OOs, SEQUENCE: 132

Calacagagtt acagtacc cc gtggacg 27

<210s, SEQ ID NO 133 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO3)

<4 OOs, SEQUENCE: 133 US 8,551,476 B2 165 166 - Continued

Gln Glin Ser Tyr Ser Thr Pro Trp Thr 1. 5

<210s, SEQ ID NO 134 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO9)

<4 OOs, SEQUENCE: 134 cgc.gcaagtic agagcatcga cacct attta aat 33

<210s, SEQ ID NO 135 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO9)

<4 OOs, SEQUENCE: 135 Arg Ala Ser Glin Ser Ile Asp Thr Tyr Lieu. Asn 1. 5 1O

<210s, SEQ ID NO 136 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO9)

<4 OOs, SEQUENCE: 136 gctgcatcca agttggaaga C 21

<210s, SEQ ID NO 137 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO9)

<4 OOs, SEQUENCE: 137 Ala Ala Ser Lys Lieu. Glu Asp 1. 5

<210s, SEQ ID NO 138 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO9)

<4 OOs, SEQUENCE: 138 caacagagtt acagtic cc cc tict cac 26

<210s, SEQ ID NO 139 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO9)

<4 OOs, SEQUENCE: 139 Gln Glin Ser Tyr Ser Pro Pro Leu. Thr 1. 5 US 8,551,476 B2 167 168 - Continued <210s, SEQ ID NO 140 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO2)

<4 OOs, SEQUENCE: 140 tctggagata aattggggga taaatttgct tcc 33

<210s, SEQ ID NO 141 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR1 (LiO2)

<4 OOs, SEQUENCE: 141 Ser Gly Asp Llys Lieu. Gly Asp Llys Phe Ala Ser 1. 5 1O

<210s, SEQ ID NO 142 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO2)

<4 OOs, SEQUENCE: 142

Caagatagga agcgt.ctict c a 21

<210s, SEQ ID NO 143 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR2 (LiO2)

<4 OOs, SEQUENCE: 143 Glin Asp Arg Lys Arg Lieu. Ser 1. 5

<210s, SEQ ID NO 144 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO2)

<4 OOs, SEQUENCE: 144

Caggcgtggg acaccalacac ttggtc 27

<210s, SEQ ID NO 145 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: WL-CDR3 (LiO2)

<4 OOs, SEQUENCE: 145 Glin Ala Trp Asp Thr Asn Thr Val Val 1. 5

<210s, SEQ ID NO 146 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Murinae gen. sp.